Ann. appl. Biol. (1962), 50, 451-460 With I plate Printed in Great Britain

Yellow-net virus of sugar beet I. Transmission and some properties

BY MARION WATSON Rothamsted Experimental Station, Harpenden, Herts.

(Receiced 6 February 1962)

SUMMARY

A virus found in sugar beet in England causes similar symptoms to the Californian virus called yellow-net (YNV) by Sylvester (1948). It persists in the feeding vectors for at least 7 days. The minimum feeding time needed for aphids to acquire virus from infected leaves was 6 hr. and the minimum needed to acquire and transmit was 12 hr., but ability to transmit was greatly increased by increasing feeding time up to 2 days on infected and 24 hr. on healthy plants. These properties agree with those of the American virus. Experiments using different aphid numbers feeding for constant times suggested that susceptibility of sugar beet to infection varies and this may account for some of the delay in infecting healthy plants.

YNV was accompanied in beet by a second virus, yellow-net mild yellows (YNMYV). Y N M W did not infect Nicotiana clevelandi Gray or N. bidlovia, which are susceptible to YNV, but from which YNV could not be recovered by aphids. Neither virus is transmissible by inoculation with sap from infected plants. It is suggested that YNMYV is a ‘carrier’ virus, without which YNV cannot be transmitted. Possibly it combines with YNV to form an aphid-transmissible combination, but it may merely help YNV to invade sugar beet.

YNMYV causes similar symptoms and has similar properties and host range to other viruses that cause mild yellowing diseases of sugar beet, including avirulent strains of beet yellows virus ( B W ) , Irish mild yellows (IMSV) and ‘41 yellows’ (41W). No such virus has been reported associated with YNV in America, and possibly it is peculiar to the British isolate.

I N T R O D U C T I O N

A virus resembling beet yellow-net virus described from California by Sylvester (1948) was isolated by Dr R. Hull from sugar beet in Lincolnshire in 1950 (Watson, 1951 and Rothamsted Reports, 1951, 1954). It was transmissible by Myzus persicae Sulz., as a persistent virus (Watson & Roberts, 1939), but not by manual inoculation of sap from infected plants. Its main difference from the Californian virus seemed to be that aphids took longer to transmit and did so infrequently and erratically, but this is probably an expression of the unusually great variability in transmissibility on which Sylvester commented (1958); his virus was transmitted easily in 1949 but much less so in 1958. Seasonal and possibly local variations in growing conditions may account

452 MARION WATSON for some of this variability, but much seems to be an intrinsic property of the virus. Experiments using different varieties or breeder’s pure lines of sugar beet, or aphids from different wild sources, did not make results more consistent and attempts to select more readily transmitting virus strains all failed. The results given here were accumulated over several years at favourable times for transmission.

M A T E R I A L S A N D M E T H O D S

All YNV was from the original plant found by Dr Hull; the aphids used were from stocks maintained continuously in the glasshouse, and the methods of handling them were as described previously (e.g. Watson, 1940). Aphids were usually fedon detached leaves that had only recently developed yellow-net symptoms. Leaves of old plants with well-developed symptoms, or completely chlorotic leaves, were not good sources of virus. Aphids fed less readily and produced fewer young when fed on them than they did on corresponding leaves of healthy plants. In this paper ‘corresponding leaves’ means leaves of the same order of phyllotaxy from plants of the same age. Unless otherwise stated they were used in all comparative experiments. The variety of sugar beet used was Kleinwanzleben E; plants were usually infected at the stage when the first true pair of leaves was emerging. Other viruses used were from stocks maintained in the glasshouse (Watson, 1951). They were: ( I ) two strains of SBYV, the type strains, and SBYNV which causes severe veinal necrosis (etch); ( 2 ) Irish mild yellows, IMSV (Clinch & Loughnane, 1948) which resembles the mild yellowing viruses found in Great Britain (Watson, 1946, 1951; Russell, 1958); (3) 41YV, the aphid- and seed-transmitted yellowing virus found in (but not confined to) Family 41 sugar beet, a breeder’s pure line developed in Eire in 1946 (Clinch & Loughnane, 1948; Watson, Hull & Hartsuijker, 1949; Watson, 1951).

Symptoms of Yh’V and its relation to other yellowing airuses of sugar beet Plants infected with YNV exhibit a yellow or whitish network along the veins over

all or part of the leaves (Pl., fig. I A). The vein-bands may coalesce until nearly all the leaf is chlorotic (Pl., fig. 2). Leaf texture is smooth and soft, not brittle as with BYV infection. Nevertheless, the first appearance of vein clearing can be confused with the onset of ‘etch’ (Clinch & Loughnane, 1948), a veinal symptom caused by virulent strains of BYV (e.g. BYNV, Watson, 1951; Watson & Russell, 1956) as it may have been by Klinkowski & Schmelzer (Klinkowski, I 952) ; their illustration, labelled ‘ Gelbnetzvirus’, is probably of etch (SBYNV) which causes pitting and superficial necrosis of the tissues around the veins, often temporarily accompanied by bright veinal chlorosis (Pl., fig. 4). Mild yellowing viruses of sugar beet, such as IMSV, cause no etch but only yellowing and brittleness of the older leaves, which is also caused by B W .

YNV was inoculated to sugar beet in combination with BYV and IMSV in the following ways :

(I) Aphids fed separateIy on YNV- and BYV-infected plants were then fed together on healthy test plants.

Yellow-net virus of sugar beet 453 ( 2 ) Aphids with BYV were fed on plants showing early symptoms of infection by

(3) Aphids with YNV were fed on plants showing early symptoms of infection by

(4) Aphids were fed on leaves of plants infected by treatments (2) and (3). Treatment (I). When groups of aphids were fed separately on YNV- and BYV-

infected plants and then together on test plants, etch symptoms caused by BYV appeared normally after 10-12 days, and yellow-net 10-20 days later. Normal yellow-net symptoms appeared in control test plants 14-2 I days after inoculation, starting with veinal chlorosis visible on the youngest leaves as in PI., fig. 3A. When combined with BYV, YNV caused modified symptoms that appeared first on fully expanded leaves, with irregular, broken veinal-chlorosis (Pl., fig. 3 B).

Treatment (2). When BYV was introduced to plants showing yellow-net symptoms, most plants became infected, but etch symptoms were often difficult to distinguish and the presence of B W had to be confirmed by transmission to healthy plants.

Treatment (3). When YNV was introduced to plants showing beet yellows, yellow- net symptoms were again delayed for a few days, and were much modified, usually starting with small chlorotic spots or flecks on well-developed leaves (Pl., fig. IB). Later, in some plants, more recognizable yellow-net symptoms developed, but often the flecks remained, or disappeared leaving the plants apparently infected with B W only.

Inoculation of YNV to IMSV infected plants was more difficult because symptoms of Irish mild yellows take several weeks to develop and YNV does not easily infect older plants. On two occasions IMSV-infected plants became infected with YNV and produced apparently normal symptoms for their age, but further work was impracticable.

BYV-infected plants were therefore susceptible to YNV infection and vice versa but the symptoms caused by the challenging virus were modified.

The nature of the interference is not understood; sap from YNV-infected plants does not precipitate with antiserum prepared against B W , nor does it react with sera of rabbits injected with such sap. Six weekly intraperitoneal injections failed on two occasions to produce serum with any ability to precipitate specifically, and intra- muscular injections of partially clarified saps plus adjuvant, also failed. Attempts to get serological reaction with IMSV have similarly failed. No particles resembling those of BYV (Leyon, 1951; Horne, Russell & Trim, 1959) have been observed in electron microscope preparations from YNV or IMSV infected plants. The particles described by Bjorling & Ossiannilsson (1958) have not been seen, but their method of extracting sap from infected leaves has not been used.

YNV.

BYV and others showing recognizable symptoms of infection by IMSV.

Yellow-net mild yellows virus (YNMYV) Plants infected with YNV developed not only the symptoms described above, but

the older leaves also became yellow and thickened as when infected by IMSV and avirulent strains of BYV. Aphids fed on these leaves sometimes transmitted YNV, but more often a virus that caused the yellowing without the yellow-net, which I shall call yellow-net mild yellows (YNMYV). This has been transmitted repeatedly without

454 MARION WATSON YNV recurring. Symptomatologically it is indistinguishable from other mild yellowing viruses of sugar beet (BYMYV, IMS, 41YV) and it has the same host range, which differs from that of YNV (see Table I).

Table I. Plant hosts of beet yellows, yellow-net, yellow-net mild yellows, and Irish mild yellows viruses

Viruses tested using infective iZfyzus persicae

S BYV YNV Y N M W IMSV 41 w Beta

B. vul.qaris + + + + + B. maritima + + ?

c. annaranticolor + C. album + C . quinoa + C. capitatum + C . foliosum + .V. clmelandi Gray -4- + N. bidloria + +

Chenopodium - - F - - - - - - - - -

- - - - -Yicotiana

- - - - - -

- - Gomplitena globosa + + Host susceptible; - host apparently immune; . not attempted.

Host range Sylvester tested twenty plant species including several of Chenopodiaceae for sus-

ceptibility to YNV, but found only Beta aulgaris susceptible. Table I shows the species tested for susceptibility to B W , YNV, YNMYV and two mild yellowing viruses of sugar beet. Most species of Chenopodium tested were immune to all except BYV. Nicotiana clevelandi and N . bidlovia were susceptible to BYV and YNV, but not to the mild yellowing viruses. The symptoms caused by YNV in these two species are very conspicuous and resemble those in sugar beet (Pl., fig. 5 ) .

Table 2 . Recozery of yellow-net virus from Nicoliana clevelandi, N. bidlovia and sugar beet

Host to which transmitted

Sources of YNY Sugar beet S. clevelandi N . bidlooia Total

Sugar beet 109'204 27'64 I 018 I 1461309 R'. clm elandi 2'133 I,ZO 0/20 3'173 N. bidlovia 1 . 5 1 0,20 oi20 I '91

Isolation of YiVV from Nicotiana spp. I"\' was never transmitted with certainty from infected N . clevelandi or N .

bidloe,ia. Table 2 shows the results of all experiments in which attempted transmissions from the Nicotiana spp. were compared with successful ones from sugar beet. Usually many more aphids were used on the Nicotiana hosts than the ten per plant used on sugar beet, but over the whole period only four transmissions were recorded; these were probably contaminations, because the infected sugar beet contained YNMYV

Yellow-net virus of sugar beet 455 which has never infected the Nicotiana spp. The aphids feed well on the Nicotiana hosts and readily isolate BYV from plants jointly infected with it and YNV. The Nicotiana hosts were also moderately susceptible, for 42 yo of N . clevelandi and 25 yo of N . bidlovia became infected compared with 52 % of sugar beet.

The results suggest that YNV and YNMYV may affect one another as Smith (1946) suggests tobacco mottle virus and tobacco vein-distorting virus do, namely, that the one enables the other to be transmitted by the aphid vectors. YNMYV may be acting as a carrier for YNV, which seems unable to be isolated from the hosts that YNMYV cannot infect.

Persistence in the feeding vectors In an experiment repeated on four occasions, aphids were fed 48 hr. on leaves

showing yellow-net symptoms and then removed in groups of ten to five test plants, from which they were transferred to fresh healthy plants for 6 days. The first two and the last of the serial feeds were of 24 hr., but on different occasions one or other of the later feeds was of 48 hr. : the last transfer was always on the sixth day. Loss and death of the aphids caused the numbers of plants infected to decline rather rapidly with time, but loss of infectivity from individual aphids was slow, and many remained infective until they died (Table 3). This agrees with Sylvester's (1949) results.

Table 3 . Infection caused during successive feeds on healthy plants Days after leaving infected plants

I 2 3 4 5 6 , >

Y N M W YNV No. of plants infected 16 I1 4 4 3 2

7 3 I 2 I 0

No. of plants tested 20 20 15 I 0 9 I2 No. of aphids recovered per plant I 0 8.5 5'2 3'7 3'0 2'0

Probability of infection by a 0.148 0.091 0.070 0.129 0 1 1 2 0.087 single aphid 0.042 0.019 0.014 0.059 0.057 o

EfJect of varying feeding time on infected and healthy plants An experiment was repeated five times in which aphids were fed for 6, 24 or 48 hr.

first on leaves showing yellow-net symptoms and then on healthy test seedlings; for each combination of treatments five plants were used on each occasion, and six aphids per plant. Table 4A shows how many plants out of twenty-five tested for each com- bination of treatments developed symptoms. The data are given for YNV and YNMYV separately, assuming that YNMYV is always present in YNV-infected plants.

Table 4B shows the data for YNMYV converted to angles, giving a statistical estimate of significance. This was not possible for YNV because the figures were too small and variable, but the totals seem to follow the same trend as for YNMYV. Differences significant at the 5 % level are about 28 times the appropriate standard error given in the table.

TableqC shows the total numbers of plants infected on each occasion and gives some idea of the variation, especially with YNV, between repetitions of the experiment.

456 MARION WATSON

Table 4. Transmission of yellow-net and yellow-net mild yellows viruses by aphids f ed for different times on infected and healthy plants

-4. iVumbers of plants infected out of twenty-jive tested for each combination of treatments

Yellow-net virus Yellow-net +YNMYV Times on 7-

infected plants.. . 6 hr. 24 hr. 48 hr. Total 6 hr. 24 hr. 48 hr. Total

Times on test plants 6 hr. I 3 6 10 5 I2 11 28 2+hr. 3 3 1 1 17 14 13 20 47 48 hr. 6 7 I 2 25 13 16 19 48

Total 1 0 13 29 5 2 32 41 50 123

B. Yellow-net + Y N M Y V data converted to angles Times of feeding on infected

Times of plants feeding on

healthy plants 6 hr. 24 hr. 48 hr. S.E. Mean S.E.

6 hr. 21'0 43.8 38.3 8.35 34'4 - + 4.82 24 hr. 48.7 43'2 69.0 * 8.35 53'6 & 4.82 48 hr. 49'0 54'3 66.7 k 8.35 57'3 & 4.82

Mean 39'5 47. I 58.0 k 4.82 482 zt: 2.78

C . Variation in transmission between the dzfferent occasions Occasions

I 2 3 4 5

Yellow-net 1 2 26 I 13 o 52 Tellow-net+YSMI?.' 27 34 14 29 19 123

Difference 1 5 8 1 3 16 19 71

The numbers of plants infected increased at a fairly constant rate with increasing feeding time on infected plants, which could reflect the times taken by different individual aphids to acquire virus, or to accumulate enough to cause infection. In contrast, infectivity increased with increasing times on healthy plants only between 6 and 24 hr., and not between 24 and 48 hr., even when the time on infected plants had been short. Sylvester (1958) suggests that YNV, unlike BYV, has a latent period in the vectors and much of his data support this. In particular aphids fed 24 or 48 hr. on infected leaves infected fewer plants immediately after leaving the source of infection than they did of a fresh batch of plants fed on 24 hr. later. On the other hand in both Sylvester's and the present experiments infectivity of aphids fed for short times on infected plants increased insignificantly even after prolonged feeding on healthy plants, and it is obvious that the quantity of virus acquired limits their infectivity, however long they are tested.

Transmission by ztarying numbers of aphids Earlier (Watson, 1936) I showed that the distribution of infection caused by groups

of I , j, 10 or 20 Myzus persicae transmitting henbane mosaic virus closely fitted

Yellozu-net virus of sugar beet 457 maximum likelihood values calculated on the assumption that the infections were local and independent. If plants could be infected by accumulation of subinfective doses from different aphids large aphid groups would infect more plants than expected on this assumption, and small groups would infect fewer. With henbane mosaic small aphid groups infected more plants than expected and large groups infected fewer. The discrepancy was not enough to affect the significance of the approximation between the observed and expected values, but it was consistent, and was attributed to variation in susceptibility of the host plants, some needing more aphids to infect them than others.

1956: YNV Y N M W

Table j. Effect of varying numbers of aphids per plant in transmission of yellow-net and yellow-net mild yellows viruses

Distribution of infection among 120 plants r

I aphid 5 aphids 10 aphids 20 aphids * * r - - h - 7 *

O.V.*M.L.V. O.V. M.L.V. O.V. M.L.V. O.V. M.L.V. P x2 2.0 1.0 4.0 4'6 8.0 8.7 - - 0.025 1.19

0.052 5.03 5.0 2.0 7.0 9.3 16.0 16.5 - -

1961 : Y NV ( I 20 plants) 3.0 1.0 6.0 47 7.0 8.3 13.0 146 0,033 5.29

Y N M W (I 20 plants) 5.0 2.3 13.0 10.0 18.0 16.6 19.0 24.0 0.078 12.63 Y N M W (90 plants)+ 5.0 3.1 13.0 12.5 18.0 19.8 - - 0.102 1.81

* O.V. = observed value; M.L.V. = maximum likelihood value (assuming that infections are local and

YNV (90 plants)? 3'0 1.1 6.0 5.2 7.0 9.4 - - 0.037 4'45

independent); P = probability of infection by a single aphid; t = excluding results for twenty aphids.

Table 5 shows the effect of feeding aphids 48 hr. on leaves showing yellow-net symptoms and then distributing them in a random manner so that different groups of plants receive different numbers of aphids. The experiment was repeated eight times in 1956 using I , j, or 10 aphids evenly distributed among fifteen plants, and six times in 1961, using I , 5 , 10 or 20 aphids distributed among twenty plants, because it was thought that ten aphids might not be infecting all possible plants.

As with henbane mosaic the data fit maximum likelihood values calculated on the assumption that infections are local and independent. Also, as with henbane mosaic, there are consistently more infections by one aphid and fewer by ten or twenty aphids than expected, but the discrepancies are not significant according to the x 2 test for goodness of fit, except for YNMYV in 1961, when twenty aphids gave far fewer than their expected number of infections. When the maximum likelihood values were cal- culated on the same data but omitting the twenty aphid groups, the fit was again very good, suggesting that ten aphids were sufficient to infect with Y N M W most plants that were capable of becoming infected in that experiment.

The sugar-beet plants used in these experiments probably varied in susceptibility to YNV and YNMYV. Some needed more virus or needed to have virus introduced to more sites before initiation of infection than others. Aphids transmitting the yellow-net complex of viruses may infect several times, the frequency increasing with the amount of virus they have acquired, which obviously increases with time of

458 MARION WATSON feeding on the infected plants. The effects of increasing feeding times on both infected and healthy plants could probably, therefore, be accounted for without postulating an incubation or even a latent period, if these have any biological signi- ficance in the transmission of the virus.

If the latent period is merely the time taken for virus to be acquired and to reach the salivary glands, combined with the mathematical probability that an infective dose of virus will reach a susceptible plant-site, there seems no reason to suppose that YNV possesses one but BYV does not. However YNV is more likely than BYV to multiply in the aphids, because it is much more persistent. YNV but not B W was transmitted from aphid to aphid by blood transfusion (Harrison, 1958) and YNV passes through the moult (Sylvester, 1948) whereas B W does not (Watson, unpublished data). So there may be fundamental differences between them, which makes it surprising that they interfere with each other’s multiplication in plants.

D I S C U S S I O N

There seems good evidence that the YNV found in England is the same as the virus from California. The quantitative differences in results obtained with the two are probably expressions of its intrinsic variability. Sylvester (1949, 1958) observed that transmissibility estimated from the speed and efficiency with which aphids acquired and transmitted virus was five times greater in 1941 than in 1959; he also mentioned variability between occasions. The two viruses cause similar symptoms, have similar host ranges and behave similarly in their vectors. Transmission of both increases with feeding times on infected and healthy plants, but there seems no clearly defined period when aphids that have acquired virus are unable to transmit.

The existence of the carrier virus, YNMYV, has not been reported previously; possibly it is a peculiarity of the British isolate of YNV. Neither of the components of the yellow-net virus-complex is sap-transmissible, and YNV can be separated from YNMYV only by aphid inoculation to the Nicotiana spp. The failures to isolate YNV from these and to obtain isolates in sugar beet that do not yield YNMYV are rather strong presumptive evidence of a carrier-type relationship, but being mainly negative are not proof. Even accepting the evidence at its face value there are two possibilities. One is that YNMYV is essential for aphids to transmit YNV, the other is that YNMYV assists YNV to invade sugar beet. If YNV could not invade until YNMYV started to multiply, and was otherwise inactivated or lost, some of the variability of results with it might be explained. However, in other examples of carried persistent viruses, e.g. tobacco mottle virus (Smith, 1946) and carrot motley dwarf virus (Watson, Rothamsted Report, 1961) one component of the complex is sap-transmissible, and able to invade some of its hosts by this means; only transmission by aphids requires the presence of the carrier.

YXV has been mentioned in European literature by Bjorling & Ossiannilsson (1958), Tilinkowski (1952, 1958) and others but there is little information about where it has been found naturally infecting sugar beet. Bjorling’s virus was stated to have been experimental material obtained from England. The virus seems not to have established itself as a pathogen of sugar-beet crops in Britain, possibly because of its difficult and

Yellow-net virus of sugar beet 459 erratic transmission, or because the first introductions were too few for it to become established.

REFERENCES

BJORLING, K. & OSSIANNILSSON, F. (1958). Investigations on particles found in plants infected with beet yellow-net virus. K. Lanthkshogskolans Ann. 24, 77.

CLINCH, PHYLLIS E. M. & LOUGHNANE, J. B. (1948). Seed transmission of virus yellows of sugar beet (Beta vulgaris L.) and the existence of strains of this virus in Eire. Sci. Proc. R. Dublin SOC. 24, 307.

HARRISON, B. D. (1958). Studies on the behaviour of potato leaf roll and other viruses in the body of their aphid vector Myzus persicae (Sulz.). Virology, 6, 265.

HORNE, R. W., RUSSELL, G. E. & TRIM, A. R. (1959). High resolution electron microscopy of beet yellows virus filaments. J. Mol. Biol. I , 234.

KLINROWSKI, M. (1952). Die ‘Bohnenwelke ’ der Phaseolus-Arten, die Bronzeflecken- krankheit der Tomate, die Vergilbungskrankheit und das Gelbnetz-Virus der Ruben. 2. die Deutsche Landwirtschaft, 12, I .

KLINKOWSKI, M. (1958). Pfinzliche Virologie. II . Die Virosen des europaischen Raumes. Akademie-Verlag, Berlin.

LEYON, H. (195 I). Sugar beet yellows virus. Some electron microscopical observations. Arkiv. f & Kemi, 3, 105.

RUSSELL, G. E. (1958). Sugar beet yellows: a preliminary study of the distribution and inter- relationships of viruses and virus strains found in East Anglia, 1955-57. Ann. appl. Biol. 46, 393.

SMITH, K. M. (1946). The transmission of a plant virus complex by aphides. Parasitology, 37, 131.

SYLVESTER, E. S. (1948). The yellow-net virus disease of sugar beets. Phytopathology, 38, 429. SYLVESTER, E. S. (1949). Transmission of sugar-beet yellow-net virus by the green peach aphid.

Phytopathology, 39, I 17. SYLVESTER, E. S. (1958). Latent period phenomena in transmission of sugar beet yellow-net

virus by green peach aphids. J . econ. Ent. 51, 812. WATSON, MARION A. (1936). Factors affecting the amount of infection obtained by aphis

transmission of the virus Hy. 111. Phil. Trans. B, 226, 457. WATSON, MARION A. (1940). Studies on the transmission of sugar-beet yellows virus by the

aphis, Myzus persicae (Sulz.). Proc. roy. SOC. B, 128, 535. WATSON, MARION A. (1946). The transmission of beet mosaic and beet yellows viruses by

aphides; a comparative study of a non-persistent and a persistent virus having host plants and vectors in common. Proc. roy. SOC. B, 133, ZOO.

WATSON, MARION A. (195 I). Beet yellows virus and other yellowing virus diseases of sugar beet. Rep. Rothamst. exp. Sta. 1951, p. 157.

WATSON, MARION A., HULL, R. & HARTSUIJKER, K. (1949). Yellowing disease of ‘Family 41 ’ sugar beet. Nature, Lond., 163, 910.

WATSON, MARION A. & ROBERTS, F. M. (1939). A comparative study of the transmission of Hyoscyamus virus 3, potato virus Y and cucumber virus I by the vectors Myzzls persicae (Sulz.), M. circumflexus (Buckton), and Macrosiphum gei (Koch). Proc. roy. SOC. B, 127, 543.

WATSON, MARION A. & RUSSELL, G. E. (1956). The value of glasshouse tests with seedlings in selecting plants tolerant to beet yellows virus. Ann. appl. Biol. 44, 381.

E X P L A N A T I O N O F P L A T E

Fig. I . A. Leaf from sugar-beet plant inoculated (by aphids) with yellow-net virus when about 3 weeks old. Photographed 4 weeks after inoculation. B. Corresponding leaf from sugar-beet plant infected when 10 days old with BYV and when 3 weeks old with YNV; photographed at the same time as IA.

Fig. 2. Chlorotic leaf from plant inoculated with YNV as seedling, photographed 4 weeks later.

30 App. Biol. 50

460 MARION WATSON Fig. 3A. Plant inoculated with YNV as seedling, by aphids fed on leaf containing YNV only; photographed 33 weeks after inoculation.

Fig. 3B. Similar plant inoculated by aphids fed on leaf containing YNV and BYV. Plant is smaller and YNV symptoms were delayed and modified.

Fig. 4. Leaf from sugar-beet plant infected with necrotic strain of BYV; photographed 24 weeks after infection as seedling.

Fig. 5. YNV symptoms in N. clevelandi.