Ann. appl. Biol. (1980), 94, 77-81 With 1 plate Printed in Great Britain

77

Leaf mottling of Spartina species caused by a newly recognised virus, spartina mottle virus

BY PHILIP JONES

Rothams fed Experimental Station, Harpenden, Herts, AL.5 2JQ

(Accepted 17 September 1979)

S U M M A R Y

The cause of a previously undocumented leaf mottling of Spartina species was investigated. Negatively stained preparations of sap from mottled leaves revealed flexuous particles 725 x 12 nm. Pinwheels with associated laminar inclusion bodies were observed in thin sections of affected mesophyll cells. The virus was purified from infected Spartina anglica plants and had a sedimentation coefficient s!& in 0.01 5 M borate of 150s. The virus was transmitted by inoculation of sap to healthy Spartina anglica, but not to a range of other graminaceous or dicotyledonous species tested. It was distantly serologically related to agropyron mosaic virus, but not to other viruses with similar morphology; the name spartina mottle virus is proposed.

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

Spartina anglica C . E. Hubbard is a rhizomatous perennial grass, partly submerged by most tides, which arose from the sterile primary hybrid Spartina x townsendii H. & J. Groves by chromosome doubling (Hubbard, 1968). Although the term ‘S. x townsendii sensa lato’ or a comparable term is sometimes used to include both species (Goodman, Braybrooks, Marchant & Lambert, 1969), the terms S . x townsendii and S . anglica are used in this text unless otherwise stated.

The establishment and spread of S . x townsendii and S. anglica in Britain is well documented (Goodman, Braybrooks & Lambert, 1959). Because the two species colonise coastal mud flats very rapidly, they have been planted extensively since 1907 for the stabilisation and eventual reclamation of these areas and to combat shore erosion. Since 1924, more than 175 000 plant fragments and seeds collected from Poole Harbour have been exported to over 130 sites around the world (Hubbard, 1965). Goodman et al. (1959) estimated that the total area of Spartina in Britain was 12 100 ha, and RanweH (1967) gave a world total of 25 000 ha distributed over nine countries.

Spartina marshes in Britain, Europe and N. America are used for grazing by both cattle and sheep (Ranwell, 1961; 1967) and Spartina has also been harvested for silage in Britain (Hubbard & Ranwell, 1966). It has been suggested recently that members of the genus Spartina, plants which possess the C, pathway of photosynthetic carbon fixation (Long, Incoll & Woolhouse, 1975), could be used in the breeding and development of temperate C, cereals.

No virus disease of S . anglica has been reported although the Spartina sward suffers from ‘die-back’, a patchy degeneration of undetermined cause which occurs locally on the south coast (Goodman, 1959). This paper reports the detection in Spartina plants showing leaf mottling of a virus distantly related to agropyron mosaic virus, for which the name spartina mottle virus is proposed. 0 1980 Association of Applied Biologists

78 P H I L I P J O N E S

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

Plant material Plants of S. x townsendii showing leaf mottling were collected from Southampton Water and

mottled plants of S. anglica from the Blackwater Estuary and from Poole Harbour. A clone of each species was maintained in the glasshouse by vegetative propagation. Seed from S . anglica collected at Bridgwater Bay, Somerset, was used to provide seedlings.

Electron microscopy Leaves were ground in distilled water, and the extract filtered through muslin, negatively

stained with 2% neutral phosphotungstate (PTA) and examined in a Philips 201 electron microscope.

Tissue for thin sectioning was fixed in 2.5% glutaraldehyde in 0.05 M sodium cacodylate buffer (pH 7.2) for 8 h. After rinsing in buffer, the tissue was post-fixed in 2% buffered osmium tetroxide overnight. The pieces of leaf were then dehydrated in a graded acetone series and embedded in Epon 812. Ultrathin sections were stained on the grid with saturated uranyl acetate in 50% ethanol, post-stained with lead citrate (Reynolds, 1963), and examined in the electron microscope.

Transmission tests For mechanical inoculation experiments severely mottled leaves were ground in 0.1 M phos-

phate buffer (pH 7.0) with a small amount of Celite and the homogenate rubbed on leaves of test plants.

In tests for transmission by animal vectors, aphids were starved overnight and observed to probe for 2 min on infected S . anglica plants before being transferred (five aphiddplant) to healthy test seedlings; eriophyid mites, Abacarus hystrix, were caged on infected S. anglica plants for 2 h before being transferred (3 mites/plant) to test seedlings. The length of feeding time on the test seedlings for both aphids and mites was 24 h.

Virus pur$cation Leaves and culms from infected S . anglica plants were cut into pieces 2 cm long, washed in

tap water, rinsed in distilled water to remove surface salt deposits and homogenised in an MSE Atomix at 4 "C in an extracting fluid consisting of 3 vols 0.5 M borate buffer pH 6.8, 1 vol diethyl ether and 1 vol carbon tetrachloride, with 0.2% 2-mercapto ethanol (1 g tissue: 5 ml extracting fluid). The extract was squeezed through muslin and centrifuged at 10 000 g for 15 min. The pellet was re-extracted with an equal volume of extracting fluid and the extract centri- fuged at 10 000 g for 15 min. The supernatant fluids were combined and the virus was preci- pitated by adding polyethylene glycol mol. wt 6000 to 5% (w/v) and NaCl to 2.5% (w/v) and stirring for 3 h at 4 OC. The suspension was centrifuged at 10000 g for 10 min, the pellet dispersed overnight in borate buffer (3 original extracting volume) and the virus finally collected by centrifuging at 75 000 g for 1.5 h and resuspended in 2 ml of 15 mM borate buffer pH 6.8.

The concentration of the virus in purified preparations was roughly estimated by counting particles under the electron microscope and comparing numbers with standard preparations of other viruses of similar morphology.

Serology Purified virus, estimated to contain about 1 mg/ml, was emulsified with an equal volume of

Freund's complete adjuvant, and 1 ml of emulsion was injected intramuscularly into each hind leg of a rabbit. Bleedings were made at 2 wk intervals. Conventional tube-precipitin serological tests were used, unless stated otherwise. For comparative tests with the virus from Spartina,

Spartina mottle virus 79

antisera to morphologically similar viruses were kindly supplied by C. C. Gill (oat necrotic mottle virus), Y. C. Paliwal (agropyron mosaic virus), T. P. Pirone (sugar-cane mosaic virus, strain H), and J. T. Slykhuis (hordeum mosaic and wheat streak mosaic viruses). Electron microscope serological tests using antiserum-coated grids (Milne & Luisoni, 1977) were done with ryegrass mosaic virus and antiserum by Miss L. Torrance.

R E S U L T S

Distribution of mottled Spartina in England and Wales Plants of Spartina spp. showing leaf mottling symptoms (Plate, fig. 1) were found at a number

of sites in England and Wales (Table 1). The proportion of plants showing symptoms varied from site to site but at Poole Harbour and Lymington over 70% of plants examined had symptoms.

Table 1. Presence of infected Spartina spp. at sites in England and Waks Location Spartina spp. with leaf mottling and particles

Poole Harbour, Dorset Lymington. Hampshire Southampton Water, Hampshire Blackwater Estuary, Essex Spurn Head, Yorkshire Southport, Lancashire Dee Estuary, Clwyd Bridgwater Bay, Somerset

S. anglica, S. maritima, S. x townsendii S. anglica S. anglica, S. x townsendii S. anglica S . anglica, S. x townsendii S . anglica S. anglica S. anglica, S. x townsendii, S. glabra, S. alternifolia, Spartina sp. 2n = 76, Spartina sp. 2n = 92

Electron microscopy Extracts from mottled and unmottled leaves from each site were negatively stained and

examined by electron microscopy. Flexuous filamentous particles (Plate, fig. 2) were observed in preparations from mottled leaves but not in those from unmottled leaves. Measurements on 132 particles gave a mean length of 725 nm (S.E. of mean = 2.6) and a width of 12 nm. The particles were fragile and fragmented after a short time in PTA.

In thin sections of infected S. anglica, ‘pinwheels’ with associated laminar bodies (Plate, fig. 3) were frequently seen in mesophyll cells, confined to the yellow areas of the leaf. The pinwheels had sigmoid arms radiating from a central core. No tubular or scroll-like aggregates were observed in association with the pinwheels and no aggregation of virus particles was observed in any of the thin sections examined.

Transmission Mechanical inoculation. The virus was transmitted by manual inoculation of leaf extracts to

20 out of 24 S. anglica seedlings: the infected plants developed mottle symptoms. The following test plants were not infected by mechanical inoculation: Agropyron repens, A rrhenatherum elatius, Avena sativa cv. Blenda, Beta vulgaris, Briza maxima, Chenopodium amaranticolor, Chloris gayana, Cortaderia argentea, Cucurnis sativus, Cynodon dactylon, Dactylis glomerata, Eragrostis tef (= E. abyssinica), Erianthus ravennae, Hordeum vulgare cv. Julia, Lolium multi- florum cv. S22, Nicotiana clevelandii, N. glutinosa, N. tabacum cv. Xanthi-nc., Oryza sativa, Panicum violaceum, Pennisetum longistylum, Petunia hybrida cv. Rose of Heaven, Phalaris canariensis, Phaseolus vulgaris cv. Prince, Phleum pratense, Setaria glauca, S . italica, Sorghum nematera, Triticum aestivum cv. Cappelle-Desprez, Zea mays cv. Dekal B.

80 P H I L I P J O N E S

Insect transmission. In 225 attempts to transmit the virus to S . anglica using aphids, Aphis fabae, Metopolophiurn dirhodum, M . festucae, Myzus persicae. Rhopalosiphum padi and Sitobion avenae failed to transmit the virus to S . anglica and M. dirhodum and R . padi failed to transmit it to Arrhenatherum elatius, Avena sativa cv. Blenda, Hordeum vulgare cv. Julia, Lolium multijlorum cv. S22, Phleum pratense, Spartina x townsendii or Triticum aestivum cv. C appelle-Desprez.

In 30 attempts the eriophyid mite, A . hystrix, failed to transmit the virus to Agropyron repens, Dactylis glomerata, L . multijlorum cv. S22 or S . anglica.

Purification The purification procedure yielded 2-4 mg of the Spartina virus from each kg of tissue. The

particles were often broken but did not aggregate. In a MSE Centriscan analytical ultracentrifuge preparations of the virus sedimented as one

component. The sedimentation coefficient (s&). determined in 0-015 M borate buffer pH 6.8, was 150s.

Serology The maximum antiserum titre (1/640) was reached 6 wk after injection; this antiserum had a

titre of 1/20 in tube-precipitin tests against purified agropyron mosaic virus but antiserum-coated grids did not react with ryegrass mosaic virus in electron microscope serological tests. In tube- precipitin tests, preparations of the Spartina virus reacted with antiserum to agropyron mosaic virus (homologous titre 1/5 12) to a titre of 1/32, but did not react with the following antisera (homologous titre in parentheses): potato virus Y (1/3000), sugarcane mosaic virus - strain H (1/5 12), oat necrotic mottle virus (1/4096), hordeum mosaic virus (1/5 12), wheat streak mosaic virus (1/512), or ryegrass mosaic virus (1/3840). The Spartina virus also failed to react with antiserum to ryegrass mosaic virus in electron microscope serological tests.

D I S C U S S I O N

The presence of long flexuous particles in mottled leaves of Spartina spp., their absence from the leaves of unmottled plants, and their transmission to healthy Spartina seedlings in which they induced similar mottling shows that they are the cause of the symptoms. There is no previous record of a virus infecting Spartina in nature, but Ford (1967) reported the susceptibility of S . pectinata (prairie cord grass) to maize dwarf mosaic virus, usually recognised to be a strain of sugar cane mosaic virus (Pirone, 1972).

The virus reported here is morphologically similar to potyviruses and, like them, induces the formation of cylindrical inclusions (pinwheels) with associated laminar inclusion bodies in the mesophyll cells of infected leaves. Potyviruses typically have aphid vectors but agropyron mosaic (AMV), ryegrass mosaic (RMV) and wheat streak mosaic viruses (WSMV), although placed by some workers in the potyvirus group on grounds of particle morphology and formation of pinwheel inclusions, are transmitted by mites.

The virus from Spartina was not transmitted by aphids or mites in the limited range of tests done but its serological relationship to agropyron mosaic virus suggests that it should be grouped with this virus and that it may have a mite vector. The serological relationship between the virus from Spartina and AMV is not close enough for the virus from Spartina to be considered a strain of AMV. This view is supported by evidence from electron microscopy. The virus from Spartina is similar to RMV in that it induces pinwheels and laminar aggregates in the cytoplasm of infected cells, whereas AMV, hordeum mosaic virus, oat necrotic mottle virus, and WSMV induce pinwheels and scrolls. Because of the distant serological relationship to AMV and lack of

Annals of Applied Biology, Vol. 94, No. 1

PHILIP JONES (Facing p . 8 1)

Spartina mottle virus 81

relationship to other mite-transmitted viruses of the potyvirus group the name spartina mottle virus is suggested.

The virus appears not to be associated with dieback of Spartina because it was found both in the presence and in the absence of this disorder. Goodman et al . (1959) attributed die-back to a combination of a fine particled substratum and impeded drainage in particular areas of marsh- land; the possibility of virus involvement was not investigated. Although the virus was not trans- mitted by any of the insects tested, the disease could be disseminated widely by infected plant fragments carried by tidal currents. The transport and planting of infected plants could also have helped the virus to spread around the English coast.

I thank Dr D. A. Govier for preparing the antiserum to the virus from Spartina, Miss L. Torrance for performing the tests with ryegrass mosaic virus antiserum, and Drs Gill, Paliwal, Pirone and Slykhuis, for gifts of antiserum.

R E F E R E N C E S

FORD, R. E. (1967). Maize dwarf mosaic virus susceptibility of Iowa native perennial grasses. Phyfo- pathology 57,450-45 1.

GOODMAN, P. J. (1959). The possible role of pathogenic fungi in ‘die-back’ of Spartina townsendii agg. Transactions of the British Mycological Society 4 2 , 4 0 9 4 1 5 .

GOODMAN, P. J., BRAYBROOKS, E. M. 8i LAMBERT, J. M. (1959). Investigations into ‘die-back’ in Spartinu townsendii agg. The present status of Spartina townsendii in Britain. Journal of Ecology 47, 65 1-677.

GOODMAN, P. J., BRAYBROOKS, E. M., MARCHANT, c. J. ~r LAMBERT, J. M. (1969). Biological flora of the British Isles: Spartina x townsendii H. & J. Groves sensu lato. Journal of Ecology 57, 298-3 13.

HUBBARD, J. c. E. (1965). Spartina marshes in Southern England. VI. Pattern of invasion in Poole Harbour. Journal of Ecology 53, 799-8 13.

HUBBARD, J. c . E. (1968). Grasses. 2nd Edition. pp. 354-361. Penguin Books, Harmondsworth, Middlesex, England.

HUBBARD, J. c. E. & RANWELL, D. s. (1966). Cropping of Spartinu salt marsh for silage. Journal of the British Grassland Society 21,214-217.

LONG, s.P., WCOLL, L. D. ~r WOOLHOUSE, H. W. (1975). C, photosynthesis in plants from cool temperate regions, with particular reference to Spartina townsendii. Nature, London 257,622-624,

MILNE, R. G. & LUISONI, E. (1977). Rapid immune electron microscopy of virus preparations. Advances in Virus Research 21,265-281.

PIRONE, T. P. (1972). Sugar cane mosaic virus. ChfIIAAB Descriptions of Plant Viruses No. 88,4 pp. RANWELL, D. S. (1961). Spartinu salt marshes in Southern England. 1. The effects of sheep grazing at

RANWELL, D. S. (1967). World resources of Spartina townsendii (sensu lato) and economic use of

REYNOLDS, E. S. (1963). The role of lead citrate at high pH as an electron-opaque stain in electron

the upper limits of Spartina marsh in Bridgwater Bay. Journal ofEcology 49, 325-340.

Spartina marshland. Journal of Applied Ecology 4, 239-256.

microscopy. Journal of Cell Biology 17,208-2 10.

(Received 3 January 1979)

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

Fig. 1. Typical symptoms on Sparrina anglica leaves collected from Arne Bay, Poole Harbour. Fig. 2. Negatively stained virus particle from mottled leaf of S. x townsendii. Bar represents 200 nm. Fig. 3. Pinwheels and associated laminar inclusion bodies (L) from a mesophyll cell of an infected leaf. The pinwheel (P) shows a central electron dense core. Bar represents 0.2ym.