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Multiplication of wheat striate mosaic virus in its leafhopper vector Endria inimica
VIKOLOGY
32, 402-105 (1967)
Multiplication
of Wheat
Striate
Vector R. C. SINHA Plant
Research Insfitufe,
Mosaic
Endria AND
Virus
in Its Leafhopper
inimical
L. N. CHIYKOWSKI
Canada Department Accepted March
of Agriculture,
Ottawa,
Ontario
16, 1967
When extracts of leafhoppers (Endria i&mica Say), that had an acquisition access period of 1 day on plants infected with wheat striate mosaic virus (WSMV), were prepared at 1 and 7 days after the start of acquisition and injected into virus-free leafhoppers, 12c/c and 61’3 of the injected insects transmitted the virus, respectively. These results indicated that WSMV concentration in leafhoppers increased during the latent period of the virus in insects. In subsequent experiments the virus was transmitted serially through 7 successive groups of leafhoppers. The calculated dilution of the virus in the seventh passage was approximately lF3. Since the starting inoculum was infective only up to dilutions of lo+, t.he virlls must have multiplied during the serial passage from insect to insect. Therefore, the findings of earlier workers t,hat infective leafhoppers (E. inimica) eventually cease to transmit WSMV to plants, cannot be interpreted as evidence against virus multiplication. INTRODUCTION
virus has been considered as evidence of absence of multiplication (Freitag, 1936; Bennett and Wallace, 193s). WSMV can be t’ransmitt,ed to virus-free leafhoppers (E. inimica) by injection with extract’s of viruliferous leafhoppers (Lee, 1963). Experiments reported in this paper were designed, using the inject’ion technique, to find whether or not WSMV multiplies in its leafhopper vector.
Wheat striate mosaic virus (WSMV) in Xorth America is transmitted by t,he leafhopper Endria i&mica (Say) (Slykhuis, 1953). The latent period of the virus in leafhoppers varied from 4-6 t,o X-24 days. The virus persisted in most, leafhoppers for several weeks but was t,ransmit,ted to plants int,ermit,tently. Several transmitt,ers failed to infect’ plants after 34 days from t)he st)art of t!hc acquisition access period although they lived for at least nnot,her 24 days. Some transmitkd the virus for longer periods but none transmitted after 72 days although they lived for another lo-20 days. A4~l increase in the acquisition access period did not prolong t,he inoculative period of an insect (Slykhuis, 1963). A long latent period and the persistJenceof a virus in insects for several weeks haw been suggested as evidence of virus multiplicat~ion in an insect (Black, 1959), whereas the decrease and eventual cessation of t,ransmission by t’he vector of curly top
MATERIALS
AND METHODS
Adult E. inintica, collected at t,he Experimental Farm, Ottawa, mere used in all cxperiment#s. Although WSMV is not present in this area, samples of the field-collect,ed leafhoppers were tested on several occasions during the course of this investigat,ion t,o ascertain that’ they were virus free. k’urthermore, during the past eight years the work in our laboratory involving field-collect’cd F:. inimica has never revealed the presence of WS;\‘IV in t’hese insects. To obtain viruliferous leafhoppers, insects were caged on wheat plarks (T&&wz durum Desf. var. Ramsey) infected with WSMV. Healt’hy wheat plank
1 Contrilnltion No. 581 from the Plant, Research of Agriculture, Institut,e, Canada Department Ottawa, Ontario. 10“
WHEAT
STRIATE
MOSAIC
VIRUS
403
were used for det’ermining the infectivity of access period. The clarified ext’racts of these test insects. During t’he incubation period of leafhoppers, prepared immediately after the the virus, in most experiments, insects were acquisition and 7 days after the start of the acquisiGon, were injected into nonvirulifermaintained on Agropyron repens (L.) plants ous leafhoppers. The injected insects were which are immune to WSRIV (Slykhuis, caged for 2 weeks on healthy A. repens 1963). In some serial passage experiments wheat, plants were used because insect sur- plants and were t,hen tested singly for 2 vival rate on them was higher than on A. weeks (1 meek on each of two successive repens plants. When wheat plants were used wheat) plants) for their ability to transmib. the insects were transferred to new plant’s The combined result,s of 2 experiments every 3 or 4 days. That inject’ed insects did showed that 7 of 57 (12%) and 3s of 62 not inoculate and reacquire virus from such (61%) insects trnnsmit,ted the virus when injected with clarified extracts prepared implants was demonstrated in the following way. Aft’er the injected insects were removed mediately after the acquisit)ion feed and at from the wheat plants, 30-40 healthy insects 7 days, respectively. These results suggest were caged on them for 4 days and then that the virus may have increased in COIImaintained on healthy plants for 4 weeks. centration in the leafhoppers between 1 and 7 days. n’one of these plants became infect’ed. All insects were maintained in a greenhouse with Serial Passage of Virus thyou!gh Leafhoppers fluctuating temperatures (24’-2K). Extracts of viruliferous leafhoppers were One met’hod to prove virus mult,iplication prepared by grinding the insects in PBS in an insect vector is to serially pass the (phosphate-buffered saline, 0.01 M potas- virus from insect to insect, by t’he injection sium phosphate, 0.15 M SaCl, pH 7.0) with technique until the dilution attained exceeds 0.1 11 glycine and 0.01 flil magnesium chlo- the dilution end point of the starting matcride used as protective factors (Brakke, rial (Maramorosch, 1952; Black and Brakke, 1956). The extracts were centrifuged at 1952). WSMV inoculum for starting serial 10,000 rpm for 10 minubes in a Spinco model passage experiment’s was prepared from leafL centrifuge in a no. 40 rotor, and the super- hoppers which had an acquisition access natants were then passed through a Rlillipore period of 3 days and then were maintained filt,er (HA 0.45 ~1, 13 mm). Detailed pro- on healthy ,4. repens plants for 4 days. Assay cedures for grinding, centrifuging, and filterexperiments showed that such inoculum ing were as described recently (Sinha and was infectious at dilutions lo-‘, 10P2, lOP, Chiykowski, 1967). Nonviruliferous insects and 10e4, but not’ 10P5. The numbers of inwere inject’ed wit’h such clarified extracts in sects that t)ransmitt,ed the virus out of those a cold room (4°C). Before injection, the ininjected with each dilution were 29/31 sects were immobilized by placing them in a (94%)), 24/30 (SO%), 16/‘0 (SO’S), A,/17 test tube immersed in an icebath. In calcu(24 ‘:‘(I), and O/21 (0 %), respectively. lating t’he dilution of the clarified extracts, it In the first passage, 18 exposed leafhoppers was assumed that a group of leafhoppers (original source) weighing 44 mg were ground weighing 1 g had a volume of 1 ml and t’hat 1 in 0.396 ml of PBS (to give a dilution of g of tissue in 9 ml of PBS equalled a l/10 10-l); the ext*ract was clarified and injected diMion. into a group of nonviruliferous Icafhoppers. The inject,ed insect’s were maintained on RESULTS healthy A. repens plants for 7 days. Then Increase of Virus Concentration in Insects some of t,hc leafhoppers were tested singly during the Latent Periocl for 7 days on wheat plants for their ability Increase in virus concentration in leaf- t’o transmit the virus, and the remainder were used for preparing the clarified extracts hoppers has been interpreted as presumptive evidence of virus multiplication in insects for t’he next, passage. This procedure was repeated every 7 days for a total of 7 pass(Black, 1941). In the following experiments ages. In each passage, t’he weight of injected adult leafhoppers had a l-day acquisition
405
SIXHA TARLE
SERI.\L VIRUS
P.\SS.ICE THROUGH
1
OF WHE.IT STRI.ITE THE LE.ZFHOPPER
Endrin
,4NL) CHIYKOWSKI
?v2os\rc: \-ECTOR
inimica
Number of insects
Experiment 11 17 15 IF 14 9 23
IF 20 18 20 20 16 36
14 13 14 15 10 8 9 Experiment
d 19 20 20 20 14 31
NCd NC 18 10 13 21
16 12 5 10 8 9
1 88 65 78 75 50 50 25
18 20 25 24 25 25 15
10-I 10-a 10-b
2 81 60 25 50 57 29
20 13 25 25 25 16 13
lo-’ 10-a 10-5 lo-’ 10-g 10-11 10-13
10-T 10-Q 10-11 10-13
n Number of insects tested singly for 7 days for their inoculativity 7 days after injection. b Number of insects that survived the 7-day test feed. c Percentage transmission was calculated from the number of insects at the beginning of the test feed. d Dash means that leafhoppers were not tested singly. NC means that, surviving insects were not counted.
insects used to prepare the inoculum was taken as a basis for making a 10-l dilution. The act’ual dilution of the inoculum in each passage was calculated in t’he manner described by Maramorosch (19.52). Single insect’s weigh about 2 mg which is at least 10 times as much as the inoculum used for their injection (about 0.0002 ml or 0.2 mg). Therefore, t,he dilution of the original inoculum in each consecutive passage increased one hundredfold. The percentage transmission by the injected insects in each passage was calculated from the number of insects caged singly at the beginning of the 7-day test feed. Some insect’s died during this period, and since the plants on which the insects died were not marked we do not know how
many insect’s transmitted the virus before they died. The results of 2 serial passage experiments showed (Table 1) that WSMV was &I1 infectious in the seventh passage. The calculated dilution of the virus in the last passage, if no multiplication had occurred, would have been 10-l”. Since the dilut,ion end point, of the original inoculum was bet,ween 1O-1 and 10P5, \VS;\IV must have mult,iplied in its vector I?. irkmica during the serial passage of t)hc virus from insect to insr& DISCUSSION
The possible increase in WSJLV conccntration during the latent period and the serial passage of t’he virus through leafhoppers provide evidence t,hat WSJIV muhiplies in its leafhopper v&or. The reason for t’he lower percentage transmission in the sevent)h passage is not known. ,I similar decrease occurred in passage 4 of experiment’ 2 but was followed by an increase in percentage transmission in passage 5. In serial passage experime& with aster yellows virus (Maramorosch, 1952), injected insects in two passages failed to infect’ plant,s, but the virus was recovered from t,hem in the subsequent passages. Altmhough WSMV multiplied in the insects, net virus t)iter seems to have dropped during the course of the seven passages. The loss of inoculat’ivity by E. inimica leafhoppers (Slykhuis, 1963) cannot be attributed to the lack of WSMV multiplication in an insect. The serial passage experiments, however, were done at intervals of 7 days, and it is possible that t,he rate of virus multiplication in insects decreases after a certain period and that WSbIV concentration may drop to a level where no transmission can occur. Decrease in virus concentraCon in leafhopper vectors has recently been demonstrated for wound-tumor and western X-disease viruses (Reddy and Black, 1966; Whitcomb et al., 1966). Our knowledge of propagative leafhopperborne viruses suggests that the virus, after reaching the blood, must pass into the salivary glands before an insect can transmit (Sinha, 1965). Anot,her possibility for loss of infectivit’y, t,hcrefore, could be that as t’he
WHEAT
STRIATE
leafhopper ages, the passage of WSMJr to the salivary glands is somehow prevented, making the insects unable to transmit even if they are viruliferous. Thus, whatever t#hereason or reasons may be for t’he loss of infectivity by leafhoppers, our results show that’ such a loss is not, necessarily incompatible with virus multiplication. Several plant viruses have been shown to mult,iply in their leafhopper vectors (Black, 1953; RTaramorosch, 1955), but, the morphology of only two such viruses, moundtumor (Brakke et al., 1954; Bils and Hall, 196%) and rice dwarf (E’ukushi et al., 1962) is known, and both arc polyhedral. The particles of WSMV are bacilliform in shape (LW, 1964). Our results provide the first crit’ical evidence that, a plant virus of this morphology is capable of mult,iplying in its vector. The particles of maize mosaic (Herold et al., 1960) and potato yellow dwarf (MacLeod et al., 1966) viruses are also bacilliform, but’ direct evidence for the multiplicat,ion of these viruses in their leafhopper vect,ors is lacking. ACKNOWLEDGMENT We wish t,o thank Mr. J. E. Lendvay-Zwickl for assist,ance in the transmission experiments. REFERENCES BENNETT, C. W., and WALLACE, H. E. (1938). Relation of t.he curly-top virus to the vector, E&fix tenellus. J. ilgr. Res. 56, 31-50. BILS, R. F., and IIALL, C. E. (1962). Electron microscopy of wound-tumor virus. Virology 17, 123-130. BL.~cK, L. M. (1941). Further evidence for multiplication of t,he aster-yellows virus in the aster leafhopper. Phytopathology 31, 120-135. BLACK, L. M. (1953). Transmission of plant viruses by Cicadellids. Advan. Virus Res. 1,69-89. BLI\CK, L. M. (1959). Biological cycles of plant viruses in insect vectors. In “The Viruses” (F. M. Burnet and W. M. Stanley, eds.), Vol. II, pp. 157-185. Academic Press, New York. BLICK, L. M., and BR.XKE, M. K. (1952). Multiplication of wound-tumor virus in an insect vector. Ph?ytoprcthology 42, 269-273.
MOSAIC
VIRUS
405
BK.\KKE, M. K. (1956). Stability of potato yellowdwarf virlls. Virology 2, 403-4iG. BR.\KKE, M. K., T-.~TTEK, A. E., and BL.~cK, J,. RI. (1954). Size and shape of wormd-tumor virlls. Brookhaz,en Symp. Biol. 5, 13i-15G. FREITAG, J. H. (1936). Negative evidence 011 m111tiplication of cculy-top virrls in the beet leafhopper. Eutetti.r lenellus. Hilgardia 10, 305-342. FTKUSHI, T., SIJJK.IT\, E., and Krnfrrlt.1, I. (1!%2). Some morphological characters of rice dwarf virus. I’irolog!/ 18, 192-205. HEHOLD, F., BEHGOLI), G. II., and WEIEEL, J. (1960). Isolation and electron microscopic demonstration of a virus infecting corn (Zeu n2a:,/,sI,.). T-irology 12, 335-347. LEE, P. E. (1983). Mechaliical transmission of wheat striate mosaic virus to its leafhopper vector Endria inimica Q:L~-. Virology 19, 88-91. LEE, P. E. (1964). Electron microscopy of inclusions in plants infected by wheat striate mosaic virus. Virology 23, 145-151. hkLEou, IX., BL.KZK, L. LI., and MO~EIL, F. H. (196G). The fine strrlcture and intracellular localization of potato yellow d&-arf virrls. T’irology 29, 540-552. ~Z.UL~OROSCH, K. (1952). Direct evidence for the multiplication of aster-yellows virus in ils illsect vector. Ph.ytopathology 42, 59-64. MIKAMOHOSCH, K. (1955). Multiplicatiou of plant viruses in insect vectors. .-t&an. I’ilus IZes. 3, 221-249. KEDDY, II. V. IL., and BLACK, L. M. (19GB). Production of wound-tumor virus and wound-tumor soluble antigen in the insect vector. Virology 30, 551-561. SINH.I, R. C. (1965). Sequential infection and distribution of wound-tumor virus in the internal organs of a vector after ingestion of virus. Virology 26, 673-686. SINHA, R. C., and CHIYBOUVSKI, L. N. (1967). Multiplication of aster yellows virus in a nonvector leafhopper. Virology 31,461-466. SLYKHUIS, J. T. (1953). Striate mosaic, a new disease of wheat in South Dakota. Phytopathology 43, 537-540. SL~KHUIS, J. T. (1963). Vector and host relations of North American wheat striate mosaic virus, Can. J. Botany 41, 1171-1185. WHITCOMB, R. F., JENSEN, D. D., and RICHARDSON, J. (1966). The infection of leafhoppers by Western S-disease virrls. II. Fluctuation of virus concentration in the hemolymph aft,er injection. Virology 28, 454-458.
32, 402-105 (1967)
Multiplication
of Wheat
Striate
Vector R. C. SINHA Plant
Research Insfitufe,
Mosaic
Endria AND
Virus
in Its Leafhopper
inimical
L. N. CHIYKOWSKI
Canada Department Accepted March
of Agriculture,
Ottawa,
Ontario
16, 1967
When extracts of leafhoppers (Endria i&mica Say), that had an acquisition access period of 1 day on plants infected with wheat striate mosaic virus (WSMV), were prepared at 1 and 7 days after the start of acquisition and injected into virus-free leafhoppers, 12c/c and 61’3 of the injected insects transmitted the virus, respectively. These results indicated that WSMV concentration in leafhoppers increased during the latent period of the virus in insects. In subsequent experiments the virus was transmitted serially through 7 successive groups of leafhoppers. The calculated dilution of the virus in the seventh passage was approximately lF3. Since the starting inoculum was infective only up to dilutions of lo+, t.he virlls must have multiplied during the serial passage from insect to insect. Therefore, the findings of earlier workers t,hat infective leafhoppers (E. inimica) eventually cease to transmit WSMV to plants, cannot be interpreted as evidence against virus multiplication. INTRODUCTION
virus has been considered as evidence of absence of multiplication (Freitag, 1936; Bennett and Wallace, 193s). WSMV can be t’ransmitt,ed to virus-free leafhoppers (E. inimica) by injection with extract’s of viruliferous leafhoppers (Lee, 1963). Experiments reported in this paper were designed, using the inject’ion technique, to find whether or not WSMV multiplies in its leafhopper vector.
Wheat striate mosaic virus (WSMV) in Xorth America is transmitted by t,he leafhopper Endria i&mica (Say) (Slykhuis, 1953). The latent period of the virus in leafhoppers varied from 4-6 t,o X-24 days. The virus persisted in most, leafhoppers for several weeks but was t,ransmit,ted to plants int,ermit,tently. Several transmitt,ers failed to infect’ plants after 34 days from t)he st)art of t!hc acquisition access period although they lived for at least nnot,her 24 days. Some transmitkd the virus for longer periods but none transmitted after 72 days although they lived for another lo-20 days. A4~l increase in the acquisition access period did not prolong t,he inoculative period of an insect (Slykhuis, 1963). A long latent period and the persistJenceof a virus in insects for several weeks haw been suggested as evidence of virus multiplicat~ion in an insect (Black, 1959), whereas the decrease and eventual cessation of t,ransmission by t’he vector of curly top
MATERIALS
AND METHODS
Adult E. inintica, collected at t,he Experimental Farm, Ottawa, mere used in all cxperiment#s. Although WSMV is not present in this area, samples of the field-collect,ed leafhoppers were tested on several occasions during the course of this investigat,ion t,o ascertain that’ they were virus free. k’urthermore, during the past eight years the work in our laboratory involving field-collect’cd F:. inimica has never revealed the presence of WS;\‘IV in t’hese insects. To obtain viruliferous leafhoppers, insects were caged on wheat plarks (T&&wz durum Desf. var. Ramsey) infected with WSMV. Healt’hy wheat plank
1 Contrilnltion No. 581 from the Plant, Research of Agriculture, Institut,e, Canada Department Ottawa, Ontario. 10“
WHEAT
STRIATE
MOSAIC
VIRUS
403
were used for det’ermining the infectivity of access period. The clarified ext’racts of these test insects. During t’he incubation period of leafhoppers, prepared immediately after the the virus, in most experiments, insects were acquisition and 7 days after the start of the acquisiGon, were injected into nonvirulifermaintained on Agropyron repens (L.) plants ous leafhoppers. The injected insects were which are immune to WSRIV (Slykhuis, caged for 2 weeks on healthy A. repens 1963). In some serial passage experiments wheat, plants were used because insect sur- plants and were t,hen tested singly for 2 vival rate on them was higher than on A. weeks (1 meek on each of two successive repens plants. When wheat plants were used wheat) plants) for their ability to transmib. the insects were transferred to new plant’s The combined result,s of 2 experiments every 3 or 4 days. That inject’ed insects did showed that 7 of 57 (12%) and 3s of 62 not inoculate and reacquire virus from such (61%) insects trnnsmit,ted the virus when injected with clarified extracts prepared implants was demonstrated in the following way. Aft’er the injected insects were removed mediately after the acquisit)ion feed and at from the wheat plants, 30-40 healthy insects 7 days, respectively. These results suggest were caged on them for 4 days and then that the virus may have increased in COIImaintained on healthy plants for 4 weeks. centration in the leafhoppers between 1 and 7 days. n’one of these plants became infect’ed. All insects were maintained in a greenhouse with Serial Passage of Virus thyou!gh Leafhoppers fluctuating temperatures (24’-2K). Extracts of viruliferous leafhoppers were One met’hod to prove virus mult,iplication prepared by grinding the insects in PBS in an insect vector is to serially pass the (phosphate-buffered saline, 0.01 M potas- virus from insect to insect, by t’he injection sium phosphate, 0.15 M SaCl, pH 7.0) with technique until the dilution attained exceeds 0.1 11 glycine and 0.01 flil magnesium chlo- the dilution end point of the starting matcride used as protective factors (Brakke, rial (Maramorosch, 1952; Black and Brakke, 1956). The extracts were centrifuged at 1952). WSMV inoculum for starting serial 10,000 rpm for 10 minubes in a Spinco model passage experiment’s was prepared from leafL centrifuge in a no. 40 rotor, and the super- hoppers which had an acquisition access natants were then passed through a Rlillipore period of 3 days and then were maintained filt,er (HA 0.45 ~1, 13 mm). Detailed pro- on healthy ,4. repens plants for 4 days. Assay cedures for grinding, centrifuging, and filterexperiments showed that such inoculum ing were as described recently (Sinha and was infectious at dilutions lo-‘, 10P2, lOP, Chiykowski, 1967). Nonviruliferous insects and 10e4, but not’ 10P5. The numbers of inwere inject’ed wit’h such clarified extracts in sects that t)ransmitt,ed the virus out of those a cold room (4°C). Before injection, the ininjected with each dilution were 29/31 sects were immobilized by placing them in a (94%)), 24/30 (SO%), 16/‘0 (SO’S), A,/17 test tube immersed in an icebath. In calcu(24 ‘:‘(I), and O/21 (0 %), respectively. lating t’he dilution of the clarified extracts, it In the first passage, 18 exposed leafhoppers was assumed that a group of leafhoppers (original source) weighing 44 mg were ground weighing 1 g had a volume of 1 ml and t’hat 1 in 0.396 ml of PBS (to give a dilution of g of tissue in 9 ml of PBS equalled a l/10 10-l); the ext*ract was clarified and injected diMion. into a group of nonviruliferous Icafhoppers. The inject,ed insect’s were maintained on RESULTS healthy A. repens plants for 7 days. Then Increase of Virus Concentration in Insects some of t,hc leafhoppers were tested singly during the Latent Periocl for 7 days on wheat plants for their ability Increase in virus concentration in leaf- t’o transmit the virus, and the remainder were used for preparing the clarified extracts hoppers has been interpreted as presumptive evidence of virus multiplication in insects for t’he next, passage. This procedure was repeated every 7 days for a total of 7 pass(Black, 1941). In the following experiments ages. In each passage, t’he weight of injected adult leafhoppers had a l-day acquisition
405
SIXHA TARLE
SERI.\L VIRUS
P.\SS.ICE THROUGH
1
OF WHE.IT STRI.ITE THE LE.ZFHOPPER
Endrin
,4NL) CHIYKOWSKI
?v2os\rc: \-ECTOR
inimica
Number of insects
Experiment 11 17 15 IF 14 9 23
IF 20 18 20 20 16 36
14 13 14 15 10 8 9 Experiment
d 19 20 20 20 14 31
NCd NC 18 10 13 21
16 12 5 10 8 9
1 88 65 78 75 50 50 25
18 20 25 24 25 25 15
10-I 10-a 10-b
2 81 60 25 50 57 29
20 13 25 25 25 16 13
lo-’ 10-a 10-5 lo-’ 10-g 10-11 10-13
10-T 10-Q 10-11 10-13
n Number of insects tested singly for 7 days for their inoculativity 7 days after injection. b Number of insects that survived the 7-day test feed. c Percentage transmission was calculated from the number of insects at the beginning of the test feed. d Dash means that leafhoppers were not tested singly. NC means that, surviving insects were not counted.
insects used to prepare the inoculum was taken as a basis for making a 10-l dilution. The act’ual dilution of the inoculum in each passage was calculated in t’he manner described by Maramorosch (19.52). Single insect’s weigh about 2 mg which is at least 10 times as much as the inoculum used for their injection (about 0.0002 ml or 0.2 mg). Therefore, t,he dilution of the original inoculum in each consecutive passage increased one hundredfold. The percentage transmission by the injected insects in each passage was calculated from the number of insects caged singly at the beginning of the 7-day test feed. Some insect’s died during this period, and since the plants on which the insects died were not marked we do not know how
many insect’s transmitted the virus before they died. The results of 2 serial passage experiments showed (Table 1) that WSMV was &I1 infectious in the seventh passage. The calculated dilution of the virus in the last passage, if no multiplication had occurred, would have been 10-l”. Since the dilut,ion end point, of the original inoculum was bet,ween 1O-1 and 10P5, \VS;\IV must have mult,iplied in its vector I?. irkmica during the serial passage of t)hc virus from insect to insr& DISCUSSION
The possible increase in WSJLV conccntration during the latent period and the serial passage of t’he virus through leafhoppers provide evidence t,hat WSJIV muhiplies in its leafhopper v&or. The reason for t’he lower percentage transmission in the sevent)h passage is not known. ,I similar decrease occurred in passage 4 of experiment’ 2 but was followed by an increase in percentage transmission in passage 5. In serial passage experime& with aster yellows virus (Maramorosch, 1952), injected insects in two passages failed to infect’ plant,s, but the virus was recovered from t,hem in the subsequent passages. Altmhough WSMV multiplied in the insects, net virus t)iter seems to have dropped during the course of the seven passages. The loss of inoculat’ivity by E. inimica leafhoppers (Slykhuis, 1963) cannot be attributed to the lack of WSMV multiplication in an insect. The serial passage experiments, however, were done at intervals of 7 days, and it is possible that t,he rate of virus multiplication in insects decreases after a certain period and that WSbIV concentration may drop to a level where no transmission can occur. Decrease in virus concentraCon in leafhopper vectors has recently been demonstrated for wound-tumor and western X-disease viruses (Reddy and Black, 1966; Whitcomb et al., 1966). Our knowledge of propagative leafhopperborne viruses suggests that the virus, after reaching the blood, must pass into the salivary glands before an insect can transmit (Sinha, 1965). Anot,her possibility for loss of infectivit’y, t,hcrefore, could be that as t’he
WHEAT
STRIATE
leafhopper ages, the passage of WSMJr to the salivary glands is somehow prevented, making the insects unable to transmit even if they are viruliferous. Thus, whatever t#hereason or reasons may be for t’he loss of infectivity by leafhoppers, our results show that’ such a loss is not, necessarily incompatible with virus multiplication. Several plant viruses have been shown to mult,iply in their leafhopper vectors (Black, 1953; RTaramorosch, 1955), but, the morphology of only two such viruses, moundtumor (Brakke et al., 1954; Bils and Hall, 196%) and rice dwarf (E’ukushi et al., 1962) is known, and both arc polyhedral. The particles of WSMV are bacilliform in shape (LW, 1964). Our results provide the first crit’ical evidence that, a plant virus of this morphology is capable of mult,iplying in its vector. The particles of maize mosaic (Herold et al., 1960) and potato yellow dwarf (MacLeod et al., 1966) viruses are also bacilliform, but’ direct evidence for the multiplicat,ion of these viruses in their leafhopper vect,ors is lacking. ACKNOWLEDGMENT We wish t,o thank Mr. J. E. Lendvay-Zwickl for assist,ance in the transmission experiments. REFERENCES BENNETT, C. W., and WALLACE, H. E. (1938). Relation of t.he curly-top virus to the vector, E&fix tenellus. J. ilgr. Res. 56, 31-50. BILS, R. F., and IIALL, C. E. (1962). Electron microscopy of wound-tumor virus. Virology 17, 123-130. BL.~cK, L. M. (1941). Further evidence for multiplication of t,he aster-yellows virus in the aster leafhopper. Phytopathology 31, 120-135. BLACK, L. M. (1953). Transmission of plant viruses by Cicadellids. Advan. Virus Res. 1,69-89. BLI\CK, L. M. (1959). Biological cycles of plant viruses in insect vectors. In “The Viruses” (F. M. Burnet and W. M. Stanley, eds.), Vol. II, pp. 157-185. Academic Press, New York. BLICK, L. M., and BR.XKE, M. K. (1952). Multiplication of wound-tumor virus in an insect vector. Ph?ytoprcthology 42, 269-273.
MOSAIC
VIRUS
405
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