Ann. appl. Bid. (1964), 54, 153-166 With 2 plates Printed in Great Britain

I53

Carrot motley dwarf and parsnip mottle viruses

BY MARION WATSON, E. P. SERJEANT AND ELIZABETH A. LENNON Rothamsted Experimental Station, Harpenden, Huts.

(Received 23 March 1964)

S U M M A R Y

Carrots that show symptoms of carrot motley dwarf contain two viruses, carrot mottle virus (CMV) and red-leaf virus (RLV). CMV cannot be manually inoculated to carrot, but can be to some other members of the Umbelliferae, as well as to some species of the Solanaceae, Leguminoseae and Chenopodiaceae. The host range of RLV is limited to the Umbelliferae, and it is not manually transmissible but was transmitted by grafting. Cavariella aegopodiae Scop. transmits RLV alone but will transmit CMV only from plants infected with both viruses. Thus aphids were unable to transmit from coriander plants manually inoculated with CMV, but after these plants were infected with RLV by aphids, virus-free aphids acquired and transmitted both viruses from them.

Aphids remain infective with both viruses for 1-2 weeks, and retain infectivity through the moult. A minimum total of about 9 hr. is needed for acquisition and transmission ; vector-efficiency increases with increasing feeding times up to several days.

The viruses causing motley dwarf become attenuated in the glasshouse after con- tinued aphid-transmission ; avirulent isolates protect their hosts against infection by virulent ones,

Infectivity of saps from CMV infected plants.was increased by extraction at high pH in the presence of a trace of Zn, and was associated in carrot with particles 30 m p in diameter. Water-phenol extracts are almost as infective as extracts in buffer, but are inactivated by 0.02 ,ug./l. pancreatic ribonuclease.

Parsnip mottle virus (PMV) resembles CMV in many ways, but also differs in some important respects. Unlike CMV it infects celery and parsnip, and it is transmitted by C. pastinacae as well as C. aegopodiae, the only vector of CMV (in combination with RLV). PMV is transmitted by aphids from plants infected with it alone, whether these plants were infected by aphids or by manual inoculation.

Carrot plants infected first with PMV and then with the motley dwarf virus complex developed symptoms of motley dwarf, but in coriander the reverse happened. CMV and PMV appear to interfere with each other’s multiplication in the hosts, but CMV is dominant in carrot and PMV in coriander. They have some properties of distantly related strains.

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

Carrot motley dwarf virus (Stubbs, 1948, 1952) is transmitted by the willow- carrot aphid, C. aegopodiae. Stubbs found that it persisted in the vector for up to 18 days,

10 App. Biol. 54

154 MARION WATSON, E. P. SERJEANT AND ELIZABETH A. LENNON and aphids needed to feed 1-2 days on infected plants to become infective. Infective aphids could infect healthy plants in feeding times as brief as 15 min. The virus was transmitted by aphids to hosts outside the Umbelliferae, including petunia, tobacco and Datura stramonium; it was not recovered from them by aphids, but was manually transmitted from one to the other. Stubbs failed to transmit motley dwarf manually to carrot, although carrot was susceptible when inoculated by aphids.

Motley dwarf of carrots in Great Britain is caused by two viruses, carrot mottle virus (CMV) and carrot red leaf virus (RLV) (Watson & Serjeant, 1964). This explains some of the behaviour described by Stubbs. The combination of carrot mottle and red-leaf viruses will be referred to as motley dwarf (disease). Motley is an appropriate enough term, as it means ‘composed of elements of diverse or varied character’ (Oxford English Dictionary) as well as variegated in appearance.

A virus from parsnip was at first thought to be the motley dwarf complex, but later shown not to be, although it causes similar symptoms in carrot. This has been given the name parsnip mottle virus (PMV).

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

The viruses causing motley dwarf can be recovered from carrot crops in most years by feeding C. aegopodiae on plants showing symptoms. New isolates were obtained from the field occasionally, because virulence and ability to be transmitted decline in the glasshouse. CMV was obtained from infected carrots by manual inoculation to Nicotiana clevelandii or Coriander. RLV was isolated from old cultures of C. aegopodiae that had multiplied for a few weeks on apparently healthy carrots.

Virus-free cultures of C. aegopodiae and C. pastinacae were obtained from willows in the spring. C. aegopodiae cultures were kept free from contamination with RLV by starting them frequently from newly born nymphs that had not fed on plants on which their mothers were reared. These nymphs were never found to be infective in the first few generations. Parsnip mottle was isolated from garden crops of parsnip, which it commonly infects.

Aphids were usually fed for 2 days on infected plants and 3 on healthy test seedlings, after which they were killed by spraying with phosdrin. Virus-free cultures were kept in insectary compartments in aphid-proof cages that were watered through sleeves.

R E S U L T S

Host range of motley dwarf Motley dwarf transmitted by C. aegopodiae from infected carrots causes symptoms

in many species of flowering plants (Table I). Most of the hosts can be infected by aphids or by manual inoculation of sap.

Aphids have become infective only by feeding on Umbelliferae that were infected by aphids (Table 2).

Some of the known hosts are difficult to infect and have few lesions, or are almost symptomless. The negative results therefore may be doubtful except with those Umbellifers that were exhaustively tested.

Family

Umbelliferae

Solanaceae

Carrot motley dwarf and parsnip mottle viruses ‘55

Table I . Host range of motley dwarf Susceptible

Daucus carota L. ssp. sativus (Hoffm.),

D . carota L. ssp. carota, wild carrot Anthriscus sylvestris (L) Bernh., cow parsley A. cerefolium (L.) Hoffm., chervil Heracleurn spondyliurn L., cow parsnip Coriandrum sativum L., coriander

Nicotiana tabacum L. var. White Burleyt N . tabacum L. var. Holmest N . clevelandii Grey N . rusticat N . xanthit N . j a v a t Petunia Datura stramonium

carrot

Leguminoseae Trifolium incarnatum L., crimson clover Phaseolus oulgaris L.t, French bean var.

Prince

Chenopodiceae Chenopodium amaranticolort C . quinoat C . album (?)t

Amaranthaceae Gomphrena globosat Cucurbitaceae - Compositae -

Not susceptible

Pastinaca sativa L., parsnip Apium duke Mill., celery (?) Petroselinum crispum

(Mill.), parsley*

N . bidlovia N . debnei

Vicia faba L., broad bean Trifolium repens L., white

Beta vulgaris, sugar beet

clover

Cucumis sativum, cucumber

Callistephus chinensis Nees, China aster

* Virus isolated by C . aegopodiae from this host was probably parsnip mottle virus. t Local lesion hosts.

Table 2. Transmission of motley dwarf, manually and by aphids, from dzfferent hosts Aphid Aphid Manual Manual

Groups of hosts as from from from from sources of infection aphid manual manual aphid

Examples : + - - ( I ) Carrot + (2) Coriander, chervil + - + +

landii, local lesion hosts - - + + ( 3 ) T. incarnatum, N . cleve-

In Nicotiana clevelandii the symptoms were variable but usually distinctive. Plants were inoculated when they had two or three leaves 1-2 in. long. The next leaves to develop showed white delicate veinal necrosis (‘etch ’), or, when slightly older, they became mottled light and dark green, and distal growth was inhibited so that the tips became ‘pinched’ as they expanded. The veinal necrosis was confined to the distal portions of older leaves. Leaves on flowering stalks were usually symptomless, but younger leaves formed in their mils were mottled or vein-banded. Inoculated leaves developed golden, slightly necrotic, local lesions.

N . xanthi, the most reliable of the local lesion hosts, developed thin broken rings of white necrosis (Pl. I , figs. I , 2). More lesions appeared when the leaves were decolorized and stained with iodine (Holmes, 193 I).

10-2

156 MARION WATSON, E. P. SERJEANT AND ELIZABETH A. LENNON Chenopodium amaranticolor and C. quinoa developed white lesions which some-

times became necrotic on C. quinoa. They differed in size, the largest in C. amaranti- color being about I mm. in diameter while in C. quinoa (Pl. I , figs. 3, 4) they were up to 2 mm. Prince beans did not always develop lesions, but when they did the lesions were very small, encompassing very few cells. Usually they had to be counted under a binocular microscope.

Trifolium incarnatum is infected easily by viruliferous aphids, but with difficulty by manual inoculation. Plants become stunted and often dark green, with very narrow light coloured vein bands that are often broken and distorted (Pl. 2, fig. 5 A).

Carrot could not be infected manually although repeated attempts were made using virus preparations infective for other hosts.

I n contrast coriander and chervil were easily infected by manual inoculation as well as by aphids. Manual inoculation of coriander caused mild symptoms with chlorotic or necrotic flecks. Some leaves of older plants became yellowed, but did not wilt. Infection by aphids caused much more severe symptoms, similar to those caused by RLV alone (Pl. 2, fig. 2A). Outer leaves developed an orange-red colour and rapidly wilted. Even mature plants developed the same symptoms and died within 2-3 weeks.

Attenuation of motley dwarf Motley dwarf isolates taken from the field often dwarfed carrots severely, but after

subculturing in the glasshouse for many months they became less virulent. An isolate (B) was taken from the field in 1959 and another (A) in 1962. Isolate A was inoculated to carrots in the field in May 1963 and recovered in September (AI). It was also propagated in the glasshouse (A2). In the autumn of 1963 average weights of ten plants 5 weeks after being infected in the glasshouse, with the three isolates, B, A I and A2 were 5.0, 2.0 and 2.9 -t. 0-21 g. A2 (Pl. 2, fig. 3 B) induced less severe leaf symptoms than A I (Pl. 2, fig. 3A), and B induced only very mild symptoms (Pl. 2, fig. 3 C). Although the worst affected plants are always selected as sources of inoculum in the glasshouse, attenuation of the virus seems invariable.

Plants infected with isolate B failed to show any further symptoms when later re- inoculated with isolate A I , although healthy plants of the same age, inoculated at the later time with A I were severely damaged. These two motley dwarf isolates therefore behave as virus-strains.

These results may explain the differences in loss of yield caused by infections made in the field in different years (Watson & Serjeant, 1964). Plants deliberately infected in the field in 1961 with the B isolate were more stunted than those infected in 1962 with the same culture of virus.

When saps from carrots infected with motley dwarf isolates A I and B were manually inoculated to Nicotiana spp., the proportion of N . clevelandii plants infected and the numbers of local lesions on N . xanthi were about the same with both isolates (Table 3). This suggests that the concentration of CMV was not altered during the attenuation of motley dwarf, but that RLV had changed. However, RLV alone does not usually stunt carrots, although the symptoms it causes can vary, so the interaction between the two is at present obscure.

Carrot motley dwarf and parsnip mottle viruses I57

Table 3. Virulence of isolates A and B Systemic infection of N. clevelandii (whole plants) and local lesions on N. xanthi.

Inoculum from carrot

Isolate A Isolate B - - Dilution of sap 1/6 1/60 1/600 116 1/60 11600

N. clevelandii (16 plants) '5 16 4 16 14 6

' Total of six half leaves inoculated. N. xanthi (local lesion)' 44 45 6 71 39 I 9

Red-leaf virus (RL V ) Red-leaf virus is easily transmitted to some Umbellifers by C. aegopodiae, as a

persistent virus. I n carrot it causes reddening or yellowing of the foliage. The symptoms range from a trace of reddening on the first leaves produced to crimsoning of the whole plant, but there is little stunting. It was not manually transmissible to N. clevelandii or any other host. Most carrots with motley dwarf show symptoms attributable to RLV. Those that do not, contain RLV which can be isolated by using one or two aphids per plant, short times of feeding, or other limiting conditions. It also occurs alone in the field in years when spread of motley dwarf is restricted. It often occurs in old experimental cultures of C. aegopodiae kept on carrot in aphid- proof cages, but CMV itself does: not. The origin of the RLV contamination in these cultures is obscure, though suspected to be a result of seed-transmission. However, aphids fed for 2-3 days on reddened or abnormal seedlings raised from seed of infected plants did not transmit virus although after 2-3 weeks multiplying on the plants they sometimes did so.

RLV is easily graft-transmissible. Healthy carrots were grafted to carrots with RLV, by cutting through the middles of the roots and crowns, and binding halves of different carrots together. The grafts took about 4 weeks to unite below soil level; nine were successful and all their healthy halves developed symptoms between the 8th and the 9th week after grafting; aphids transmitted RLV from them. In nine successful grafts between healthy carrots, no reddening of the leaves developed. None of the hosts or scions developed symptoms of motley dwarf. Saps from them did not infect N. clevelandii.

Transmission of motley dwarf by aphids Persistence in viruliferous aphids

Aphids were fed for 2 days on carrots with motley dwarf, then transferred every day to groups of ten healthy seedlings, using fifteen aphids per seedling on the first day. Three out of four replicates tested the aphids for 8 days and one for 12 days.

Although the numbers of aphids per plant declined in the ratio of about I 5 : 9 : 6 : 4 during the transfers, nearly all plants that received aphids became infected with motley dwarf, and there was no obvious decline in infectivity during consecutive test feeding. Some of the final plants in each experiment were tested for the presence of CMV by inoculating expressed sap to N . clevelandii. All tests were positive except one, in which aphids transmitted only RLV. Therefore CMV persisted almost as well in the vectors as did RLV, although alone it is not transmitted by aphids.

158 MARION WATSON, E. P. SERJEANT AND ELIZABETH A. LENNON Passage through the moult

Aphids were fed as nymphs on carrots with motley dwarf or with RLV alone. They were then placed on healthy leaves in small tubes. After moulting to adults, they were removed to healthy test plants in groups of five aphids. Fifty per cent moulted during the first and 50% during the second day after removal from the infected plants. Eleven out of twelve test carrots developed symptoms of motley dwarf and seven out of ten became infected with RLV. C M V as well as RLV is therefore carried through the moult.

Times of feeding on infected and healthy plants Aphids were fed for various times on carrots infected with virulent or avirulent

(A and B, Table 3) motley dwarf isolates, and then for the same times on six healthy test seedlings. The experiment with isolate A was repeated on four occasions and that with B on five, using five aphids per plant. The results (Table 4) were trans- formed to degrees for statistical analysis to decrease the effect of poorly determined extremes in the proportions of plants infected on each occasion. Arithmetically the mean percentage infection for isolates A and B were 25 yo, and 4.5 yo respectively.

Table 4. Effect of varying times of feeding on infected and healthy plants Mean angular transformations

Hours of feeding on healthy plants Hours feeding on < A

infected plants 3 6 I 2 24 36 Mean S.E.

0'0 - 8.8 - 17'5 8.8

- 62.9 50.9 - 53'5 30.4} 7'33

Isolate A 3 21.3 47'3

16.4 -

Mean 19.6 - 25.8 -

12

36 42.5 -

49'7 30.0 423 - Isolate B 3 0'0 0.0 7.0 0'0

6 12'0 2.8 13.0 6.0 I 2 10.0 10.0 13.0 7.0 24 6.0 15-0 13-0 397 - I 8.4

3.11 - 10'0 -

9'7 1'55 Mean 7.0 7.0 11.5 13.2 -

Neither isolate was transmitted when aphids fed 3 hr. on infected and 3 hr. on test plants, but 0.2 % (calculated value for probability of infection by a single aphid) transmitted isolate B and 3 % isolate A, in a total of 15 hr. Differences in the rates of increase for feeding times on infected and healthy plants are not significant. Aphids fed for 3, 6 and 12 hr. on plants infected with isolate B transmitted almost at random, as though the quantities of virus they received were insufficient to initiate a regular pattern of increase of infectivity.

R E L A T I O N B E T W E E N RLV A N D C M V

Stubbs (1948) suggested that C. aegopodiae failed to transmit motley dwarf virus from petunia or other non-umbellifers because the aphids did not feed long enough on them, but in our experiments aphids did not transmit after feeding on manually inoculated coriander, a favoured host. This suggested that the disease might be

Carrot motley dwarf and parsnip mottle viruses I59 caused by a complex of viruses, as in tobacco rosette disease described by Smith (1946). In this complex, tobacco mottle, which is manually-, but not aphid-transmitted, can be transmitted by aphids in the presence of vein-distorting virus which is an aphid-transmitted, persistent virus, not manually inoculable.

When coriander seedlings were manually inoculated with CMV from carrot or from N. clevelandii symptoms often developed slowly, but by the time the plants had about a dozen leaves some of them were flecked with necrosis, or were mottled or chlorotic. Even large numbers of aphids fed for a week on whole plants, or on the most conspicuously affected leaves, never transmitted virus to carrot or any other host. Aphids, first fed on carrots infected only with RLV, were fed for z days on such CMV-infected corianders (Table 5 A) after which, in groups of ten, they were moved to carrot seedlings (Table sB), five seedlings for each coriander tested, to ensure that they were capable of transmitting RLV and had not acquired CMV from the corianders. Virus-free aphids fed 3-5 weeks later on corianders infected only with CMV did not infect test groups of carrots, but those fed on doubly-infected corianders showing red-leaf symptoms, transmitted motley dwarf to carrots from about a third of the corianders tested. Manual inoculation from the test carrots (Table 5 CI, Cz) to N . clevelandii confirmed the presence of mottle virus (Table 5 D). Aphids fed on singly-infected corianders have not transmitted CMV to carrots but they could transmit it from coriander plants in the presence of RLV, whether the CMV were isolated from carrot or from N . clevelandii.

Table 5. The transmission of CMV from coriander separately inoculated with RLV B. C. D.

Aphids fed on A then on carrots

red-leaf to A C I after 21 days C z after 28-35 days N . clevelandii'

Manual Test of aphids inoculation

that transmitted A of c to

Carrot. Carrot.

A. &&& Coriander R M R M R M CMV symptoms

Healthy (20 plants

Infectedt (23 plants

not infected) 45/70 0170 31/65 016s 16/50 4 5 0 0/42

manually inoculated with CMV) 991115 o / I 1 5 131115 I O / I I 5 17/95 2 1 / 9 5 58/75

A, Original manual inoculation to coriander. B, ' Back-test ' of aphids-demonstrating that they did transmit red-leaf but did not acquire mottle. The original corianders were also tested for aphid- transmission of mottle virus, but were negative. C, Virus-free aphids fed on doubly infected coriander after red-leaf symptoms developed. Controls were also done but were negative. R, Red-leaf symptoms. M, Mottle symptoms.

Tested in groups of 3. Twenty-three of the thirty-one carrots recorded as having motley dwarf were tested ; twenty-one proved positive.

.i. Aphids fed on twenty of these before RLV inoculation did not transmit.

Properties in vitro of CMV Carrot mottle virus is unstable in vitro. Local lesions on appropriate hosts are

formed only on leaves that are not fully expanded at the time of inoculation, some- times only on the youngest leaf of a plant. Preparations inactivate during inoculation,

160 MARION WATSON, E. P. SERJEANT AND ELIZABETH A. LENNON and lesion counts vary not only between leaves of different ages, but between the first and last leaf of the same age to be inoculated. Preparations, especially purified ones, vary greatly in infectivity but an even greater source of variation seems to be the state of the local-lesion hosts. Conditions of growth as well as immediate pre- treatment with heat or darkness affect their sensitivity. The following is an incomplete account of the properties of CMV whose behaviour gives it an unusual interest because it is both a persistent aphid-transmitted virus and a manually transmitted one.

Saps from carrot infected with motley dwarf, or N . cleve2andii infected with CMV, whether manually or by aphids, cause similar symptoms in N . clevelandii and coriander, and similar local lesions on N . xanthi, Chenopodium spp. and French bean. The CMV from N . clevelandii can be re-united with RLV in coriander, and cause motley dwarf symptoms in carrot when the combination is transmitted by aphids (see p. 159). We can therefore be satisfied that the symptoms in N . clevelandii and these other hosts are those caused by the manually transmissible component of the carrot motley dwarf complex, although carrot itself is not susceptible to manual inoculation.

Saps extracted from carrots with motley dwarf infect N . xanthi and N . clevelandii when diluted to I/IOO with phosphate or tris buffer at p H 7.3, or to over 1/500 when in borate buffer. In distilled water at a dilution of 115, most of the infectivity is lost during 6 hr. at room temperature (c. zoo C.), but a little remains after 24 hr. or even 48 hr.

Attempts at purification of the motley dwarf viruses from carrots by various methods, have often, but not invariably, associated spherical particles, about 30 m p in diameter with infectivity (Pl. I, fig. 5) . Such particles could be concentrated by a second high-speed centrifugation but would then not infect, and electron micro- graphs (Pl. I , fig. 6) suggest they may have become emptied of their nucleic acid.

Particles were not seen in infective extracts from N . clevelandii nor from healthy or RLV (with one exception) infected carrots (Table 6).

Table 6. Correlation between infectivity and particles seen in electron microscope preparations of sap from different CMV-infected hosts

Numbers of preparations

Origin

With particles Without particles PP Infective Not infective Infective Not infective Total

Motley dwarf in carrot 2 1 8 4 5 38 Motley dwarf twice centrifuged 0 2 0 0 2 Mottle in N , clevelandii 0 0 0 8 8 Red-leaf in carrot 0 I 0 I 0 I1 Healthy carrots 0 I 0 1 0 I1

We attempted to find out whether mottle virus exists in carrots and in N . clevelandii as nucleic acid protected by protein, as free nucleic acid, or as a mixture of both.

High pH, traces of heavy metal such as zinc, or bentonite are independently capable of inhibiting plant ribonuclease, and it was easily determined that extraction of saps

Carrot motley dwarf and parsnip mottle viruses 161 from both N. clevelandii and carrot in borate buffer at pH 9~1-9-4fzinc (tr.), preserved their infectivity better than extraction in tris or phosphate buffers, even at high pH (Table 7).

Table.7. Effect of different bugers used at digerent p H levels on infectivity of extracts from CMV-infected carrot and Nicotiana clevelandii

Buffers

Extracted Local Phosphate Tris Tris Borate + Zn Borate from" lesionst on PH 9.4 PH 9.6 PH 7'5 PH 9'3 PH 9'4

N. clevelandii N. xanthi 60 7 I9 68 47 Bean 14 4 5 84 I 2

Carrot N. xanthi 29 81 56 44' 178 C. quinoa 31 59 42 190 -

* 115 dilution by weight in buffer. t Total on 8 half-leaves

I n other experiments saps from N . clevelandii gave mean log. differences of 0.84 2 0-245 and 0.62 i: 0.1 39 for increase in lesion counts on leaves of N. xanthi and C . quinoa inoculated with saps extracted in borate buffer with Zn compared with those extracted in phosphate buffer at pH 7.3 (PI. I , figs. 2, 3, 4). Saps from carrot gave a mean log. difference of 0.55 0.195, in favour of extraction at high pH. These log. differences represent sevenfold to ninefold increases, and suggest that mottle virus could be present in carrot and N . clevelandii as nucleic acid. Our inability to find particles in infective saps from N. clevelandii could also support this idea.

Infective nucleic acid can be isolated from both carrot and N. clevelandii by extrac- tion with water-saturated phenol. The infectivities of water-phenol extracts were similar to those of saps extracted in pH 7.5 buffer (Table 8). Pancreatic ribonuclease at 0.02 ,ug./l. almost completely inactivated water-phenol extracts, and had a small effect on extracts in buffer alone. Carrot or N. clevelandii saps extracted in bentonite at 30 g./l. in buffer at p H 7.3, using the method described by Kassanis & Welkie (1963), were no more infective than saps extracted in the buffer alone (Table 9). If there had been free nucleic acid in the saps, bentonite would be expected to protect it. This result therefore suggests that free nucleic acid was not present. Possibly high pH, with zinc increases infectivity of saps by inhibiting some other enzyme than ribonuclease. Alternatively nucleic acid may be present, but bound, so that bentonite could not affect it.

Table 8 . The eflect of pancreatic ribonuclease on water-phenol extracts of saps from CMV infected carrot and Nicotiana clevelandii

Source ... ... Carrot N. clevelandii

Host ... ... N. xanthi" N . clevelandiif N. clevelandiif & & &

Dilution of sap ... 1 / 1 0 I/IOO 1 / 1 0 1/roo 1/10 I l l 0 0 Sap in pH 7-5 buffer 129 18 12/12 9/12 14/14 5/14

Same + 0.02 pg./l. RNase I 2 I 3/12 O / l Z 0114 4 x 4

Same +0.02 pg./1. RNase 61 I 1 12/12 8/12 14/14 9/14 Water phenol extract 85 '5 12/12 4/12 13/14 1/14

" Local lesions on 8 half-leaves.. t Whole plants.

162 MARION WATSON, E. P. SERJEANT AND ELIZABETH A. LENNON

Table 9. The efJect of bentonite on infectivity of saps from CMV infected plants, extracted at low pH. Local lesions on Nicotiana xanthi

Sap extracted in phosphate buffer pH 7.5

From N . clevelandii From carrot I ,

Exp. (-) +Bentonite (-) i- Bentonite

I 25 18 5 5 54 I1 87 33 109 5 1

Total 112 5 1 164 105

If mottle virus is not present in N . clevelandii as free nucleic acid, an alternative explanation for the absence of particles in our preparations from N . clevelandii is that the particles from carrot are those of red-leaf and not of mottle virus. If so RLV must be much less concentrated when alone than when multiplying with CMV, because particles have only once been seen in preparations from carrots infected with RLV alone.

Parsnip mottle virus (PMV) Dt@rences between PMV and CMV

The virus isolated from parsnips was transmitted to carrot, celery, and parsnip, by C. aegopodiae and by manual inoculation. Like motley dwarf PMV persists in the vector for at least 8 days and continues through the moult. The symptoms in carrot are sufficiently similar to those of motley dwarf to be mistaken for it, but important differences are:

(I) Although CMV and PMV have common hosts, motley dwarf does not infect parsnip or celery.

(2) Leaves of PMV infected carrots become flecked or mottled with yellow. The pinnules are pointed and twisted giving leaves a ‘stags horn’ appearance (Pl. 2,

fig. IB). The foliage does not become red. RLV cannot be isolated from carrots inoculated by aphids with PMV.

(3) When manually inoculated to N . clevelandii PMV produces variable symptoms often with a ring-like pattern. N . clevelandii is less susceptible to PMV than to CMV.

(4) The symptoms PMV causes in coriander are the same whether inoculated by aphids or manually, viz. extreme dwarfing with dark green mottle and crumpling of the leaves. There is little chlorosis and infected plants die prematurely (Pl. 2, fig. 4P).

( 5 ) Aphids can transmit PMV from manually infected coriander. (6) Infected crimson clover leaves have distinct yellow bands following the course

of the veins (PI. 2, fig. 5 B). When they are infected with motley dwarf the leaf pattern is indistinct and broken, and leaves are crumpled or deformed (PI. 2, fig. 5 A).

(7) PMV is transmitted by C . pastinacae as well as C . aegopodiae; motley dwarf only by C. aegopodiae (Table 10).

Relation between PMV and carrot motley dwarf An experiment to determine whether PMV could infect carrots already infected with

motley dwarf and vice versa illustrates some of the differences between the two viruses.

Carrot motley dwarf and parsnip mottle viruses 163

Table 10. Vector specificity of Cavariella aegopodiae and C. pastinacae in transmitting motley dwarf and P M V

Virus

Aphids (10 per plant) - Host C . aegopodiae C . pastinacae

Motley dwarf Carrot 25/25 0121

Parsnip mottle Carrot 11/15 13/15 - Parsnip 411 I

Ten carrots were infected with motley dwarf and ten with PMV. When symp- toms were fully developed after 3 weeks, five from each group and five healthy plants were inoculated by C. aegopodiae with the other virus. Most carrots containing motley dwarf viruses, whether they originally had PMV or not, showed only motley dwarf symptoms after 4 weeks. Only one of the originally PMV-infected plants, infected later with CMV, showed PMV symptoms but it yielded both viruses when tested, and eventually they all showed motley dwarf symptoms. Saps from the carrots were manually inoculated to coriander. PMV symptoms occurred among all groups of corianders inoculated from carrots that had been inoculated with PMV (Pl. 2, fig. 4, C + P, P + C, P), although the hosts from which the inoculum was derived, showed motley dwarf symptoms. Corianders that showed only symptoms of CMV were inoculated from plants singly infected with motley dwarf (Pl. 2, fig. 4 C and Table I I).

Table I I. Interaction between P M V and motley dwarf in carrot and coriander

Combination of viruses (in order of inoculation)*

Symptoms H + CMV PMV+ CMV H + PMV CMV + PMV Type of I A

inoculation Host

A. Original aphid- Carrot (5 plants) CMV 5 4 0 5 inoculations to PMV 0 I 5 0

carrots ? both 0 I 0 I

Aphid test ; two Carrot (10 plants) CMV 9 7 0 9 sources from A PMV 0 I 7 0

? 0 2 0 I

Manual inoculation ; Coriander CMV 7 3 0 5 two sources from A (10 plants) PMVt 0 I 4 I

* Second inoculation 3 weeks after first. t Not possible to detect symptoms of carrot mottle in coriander already infected with parsnip

mottle ; both viruses could have been present. H = Healthy plants; ? = not possible to distinguish, probably both.

Table 12. Transmission by Cavariella aegopodiae (10 per plant) from coriander, manually infected with CMV, CMV + P M V or P M V

Transmitted to carrot Transmitted to parsnip h

I , Viruses infecting coriander CMV CMV + PMV PMV CMV CMV+PMV PMV

Plants infected Plants inoculated 0110 4+/20 3/10

* PMV only transmitted.

164 MARION WATSON, E. P. SERJEANT AND ELIZABETH A. LENNON Aphids were fed on the manually inoculated corianders. They transmitted nothing from those showing symptoms of CMV only, but PMV from those showing PMV- symptoms, whatever the source (Table 12).

Parsnip mottle virus resembles the motley dwarf complex in transmission, and to a large extent, host range, but there seems to be only one virus concerned. This can be transmitted by aphids from manually infected coriander, and probably from carrot. It is virulent in both hosts, and there is interaction in plants between it and motley dwarf. Whether inoculated first or second, motley dwarf symptoms always dominated those of PMV in carrot and CMV was more frequently isolated even when PMV was the challenging virus. In coriander the reverse happened; parsnip mottle symptoms were dominant and PMV was the only virus transmitted. The viruses interfere with each other’s multiplication differently in the two hosts, and have some properties of distantly related strains.

D I S C U S S I O N

Carrot motley dwarf is caused by two viruses: ( I ) CMV, which is manually- transmissible but not transmitted by aphids except in the presence of red-leaf (RLV) ; and (2) RLV, a persistent aphid-transmitted virus that enables CMV to be aphid- transmitted. This ability is known with other viruses, some of the ‘persistent’ type; for example tobacco rosette (Smith, 1946), and beet yellow-net (Watson, 1962) diseases are caused by complexes of viruses. Others are non-persistent viruses such as potato viruses Y with C, and potato viruses Y or A with potato aucuba virus (Kassanis, 1961). Probably there are several mechanisms involved. Watson (1960) sug- gested that potato viruses Y and C change their genetic character as a result of multi- plying together in cells. Kassanis suggested that the presence of the auxiliary virus aggregates the assisted virus and enables it to be attached to the aphids’ stylets as a surface contaminant but there is no evidence for this (Close, 1962).

As CMV passes through the moult and persists for long periods in the aphids, such a temporary aggregation of particles, which would merely enable CMV to be picked up by aphids from plant cells when RLV was present, does not account for the whole of their transmission.

Kassanis (1962) described a ‘satellite virus’ of tobacco necrosis virus (TNV) which can only multiply in plants in the presence of TNV itself. If the viruses of the motley dwarf complex multiply in the aphids, as their behaviour by analogy with potato leaf roll virus (Stegwee & Ponsen, 1958) suggests, the transmission of CMV might be explained by RLV assisting its multiplication in aphid tissues as TNV does the satellite virus in plant tissues.

The properties of CMV obviously unfit it to survive in nature without red-leaf, unless, like potato virus C, it does so in vegetatively propagated plants. Parsnip mottle, by contrast, which has many of the properties of carrot mottle, and is also a persistent, aphid-transmitted virus, is on its own successfully disseminated by its two vector species except when it comes into competition with the motley dwarf complex in carrots (and possibly in some wild hosts).

Reciprocal inoculation experiments suggest that the two viruses may be related, or at least have a common origin. It is interesting to speculate whether CMV was

Carrot motley dwarf and parsnip mottle viruses 165 once similar to PMV, but lost its ability to be independently transmitted after joining with red-leaf, or whether PMV was once a dependent virus that has now acquired the ability to be aphid-transmitted.

I n some ways the carrot motley dwarf viruses and PMV resemble the barley yellow dwarf group of viruses. The interaction between them in plants is similar to that between different barley yellow dwarf viruses (Watson, unpublished data, and Rep. Rothamst. exp. Sta. 1962), and there is similar vector-specificity, one group of yellow-dwarf viruses is transmitted by R. padi and species from the subfamily Macro- siphini, the other group only by the Macrosiphini. In the same way parsnip mottle virus is transmitted by Cavariella aegopodiae and C. pastinacae, carrot motley dwarf only by C. aegopodiae. There are few examples of this behaviour among persistent aphid-transmitted viruses, though it seems common among non-persistent viruses, and Black (1944) has described strains of potato yellow dwarf specifically transmitted by leaf hopper species.

The virus causing lettuce necrotic yellows, recently described from Australia by Stubbs & Grogan (1963), has properties that may link it with motley dwarf and parsnip mottle viruses. It is transmitted by Hyperomyzus lactucae L. as a persistent virus from Sonchus oleraceus (sow thistle) to lettuce, but cannot be re-isolated from lettuce by aphids. It can be manually transmitted from Sonchus or lettuce to a number of hosts, some in common with the motley dwarf virus group, but lettuce is not susceptible to manual inoculation from any source. Lettuce seems to provide a host of necrotic yellows virus which combines the behaviour of crimson clover or N . clevelandii towards motley dwarf, with that of carrot. Motley dwarf can apparently be transmitted to crimson clover by aphids but recovered only manually. It can be transmitted by aphids to carrot and be recovered manually and by aphids, but carrot cannot be infected manually. It is interesting to speculate whether lettuce necrotic yellows consists, or ever consisted, of a combination of two viruses.

We thank Miss Sheila Bunyan for technical assistance and Mr R. D. Woods for the electron microscopy. We also thank D r R. H. Cammack of the Ministry of Agri- culture’s Plant Pathology Laboratory for drawing our attention to parsnip mottle virus.

R E F E R E N C E S

BLACK, L. M. (1944). Some viruses transmitted by agallian leafhoppers. Proc. Amer. Phil. SOC.

CLOSE, R. C. (1962). Some interactions between viruses when multiplying together in plants.

HOLMES, F. 0. (1931). Local lesions of mosaic in Nicotiana tabacum L. Contr. Boyce Thompson

KASSANIS, B. (1961). The transmission of potato aucuba mosaic virus by aphids from plants infected by potato viruses A or Y. Virology, 13, 93.

KASSANIS, B. (1962). Properties and behaviour of a virus depending for its multiplication on another. J . gen. Microbiol. 27, 477.

KASSANIS, B. & WELKIE, G. W. (1963). The nature and behaviour of unstable variants of tobacco necrosis virus. Virology, 21, 540.

SMITH, K. M. (1946). Tobacco rosette; a complex virus disease. Parasitology, 37, 131.

88, 132.

Thesis, Faculty of Science, University of London.

Inst. 3, 163.

166 MARION WATSON, E. P. SERJEANT AND ELIZABETH A. LENNON STEGWEE, D. & PONSEN, M. B. (1958). Multiplication of potato leaf roll virus in the aphid

STUBBS, L. L. (1948). A new disease of carrots; its transmission, host range and control.

STUBBS, L. L. (1952). Further host range and transmission studies with a virus disease of

STUBBS, L. L. & GROGAN, R. G. (1963). Necrotic yellows; a newly recognised virus disease of

WATSON, M. A. (1960). Evidence of interaction or genetic recombination between potato

WATSON, M. A. (1962). Yellow net virus of sugar beet. Ann. appl. Biol. 50, 451. WATSON, M. A. & SFXJEANT, E. P. (1964). The effect of motley dwarf virus on yield of carrots

and its transmission in the field by Cavariella aegopodiae. Scop. Ann. appl. Biol. 53. 77.

Myzus persicae (Sulz.). Entomol. exp. appl. I, 291.

Aust. J. Sci. Res. I , 303.

carrots endemic in Australia. Aust. J. Sci. Res. 5 , 399.

lettuce. Aust. J. agric. Res. 14, 439.

viruses Y and C in infected plants. Virology, 10, 21 1.

E X P L A N A T I O N OF P L A T E S

PLATE I

Fig. I. Necrotic local lesions of carrot mottle virus (CMV) on Nicoiiana xanthi. Fig. 2. N . xanthi leaf inoculated CMV. On left, borate buffer pH 9.3 with trace of ZnSO,, on right, phosphate buffer pH 7.3. Fig. 3. Local lesions on Chenopodium quinoa, phosphate buffer at pH 7-3. Fig. 4. Local lesions on C. quinoa, borate buffer pH 9.5, with trace of ZnS04. Fig. 5 . Particles associated with infectivity of partially purified extracts from carrots infected with motley-dwarf when manually inoculated to Nicotiana or Chenopodiznn hosts. Fig. 6 . Non-infective partially purified extract after a second high-speed centrifugation.

PLATE 2

Fig. I. A. Healthy carrot leaf. B. Leaf of same age infected with parsnip mottle virus (PMV). C. Leaf of same age infected with motley dwarf. Fig. 2. A. Coriander plant infected with red-leaf virus. (Compare with fig. 4A, coriander manually infected with CMV.) B. Healthy coriander of same age. Fig. 3. Carrots infected with: A, virulent motley-dwarf isolate from the field; B, the same isolate after 5 months’ propagation in the glasshouse; C, an originally virulent isolate after 2

years’ propagation in the glasshouse. Fig. 4. Coriander manually inoculated with saps from carrots infected by aphids with: C, carrot motley dwarf (CMV symptoms); C+P, carrot motley dwarf first, then parsnip mottle virus (PMV); P + C, PMV first then motley dwarf; P, PMV only. Fig. 5. A. Leaf from crimson clover plant infected by aphids with motley dwarf. B. Leaf from crimson clover plant infected by aphids with PMV.

The Annals of Applied Biology, T"ol. 54, No. 2 Plate I

The Annals of Applied Biology, VoI. 54, No. 2

MARION WATSON, E. P. SERJEANT AND ELIZABETH A. LENNON

Plate 2