14
Ann. appl. Biol. (1990), 116, 73-86 Printed in Great Britain 73 Seed-borne cucumber mosaic virus infection of subterranean clover in Western Australia By R. A. C. JONES and S. J. McKIRDY Department of Agriculture, Baron-Hay Court, South Perth 6151. Western Australia (Accepted 19 September 1989) Summary In Western Australia, infection with cucumber mosaic virus (CMV) was widespread in all three subspecies of subterranean clover (Trifolium subterraneum) growing in plots belonging to the Australian National Subterranean Clover Improvement Programme. Seed-borne CMV was detected in seed harvested in 1984-1986 of 18/25 cultivars from two collections of registered cultivars; seed transmission rates ranged up to 8.8%. Seed samples from CMV-inoculated plants of 11 cultivars transmitted the virus to 0.5-8.7% of seedlings. Seed transmission rates greater than 5% were obtained only with cvs Enfield, Green Range and Nangeela. CMV was not detected in seed harvested in 1975-1981 from one of the registered cultivar collections, in 17 commercial seed stocks from 1986 or in a survey of subterranean clover pastures. Symptoms in subterranean clover naturally infected with CMV included mottle, leaflet downcurling and dwarfing but severity varied with cultivar and selection. CMV isolates from different sources varied in virulence when inoculated to subterranean clover; two (both from subterranean clover) were severe, two moderate and three (including one from subterranean clover) mild. In pot tests, CMV decreased herbage production and root growth (dry wts) of cv. Green Range by 49% and 59% respectively. In spaced-plants growing in plots, CMV decreased herbage production and root growth of cvs Green Range and Northam by 59-63070 and seed production of cv. Green Range by 45%. In rows sown with infected seed, aphid spread increased infection levels to 75% in cv. Green Range and 44% in cv. Esperance and losses in herbage production of 42% and 29% respectively were recorded. CMV isolated from subterranean clover included isolates from both serogroups. Key words: Virus, seed, pasture, clover, collection, losses, infection Introduction Subterranean clover (Trifolium subterraneum) is well adapted to growing in self-regenerating pastures in medium-high rainfall (> 350 mm annually) districts of the south west of Western Australia which has a predominance of sandy soils and a Mediterranean climate. Sowing of subterranean clover in the region began in the 1930s and increased dramatically in the 1950s and 1960s, the area sown reaching about 7 million hectares by the early 1970s (Gladstones, 1975; Gladstones & Collins, 1983; Collins & Gladstones, 1984; Collins, Francis & Quinlivan, 0 1990 Association of Applied Biologists

Seed-borne cucumber mosaic virus infection of subterranean clover in Western Australia

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Page 1: Seed-borne cucumber mosaic virus infection of subterranean clover in Western Australia

Ann. appl. Biol. (1990), 116, 73-86 Printed in Great Britain 73

Seed-borne cucumber mosaic virus infection of subterranean clover in Western Australia

By R. A. C. JONES and S . J. McKIRDY Department of Agriculture, Baron-Hay Court,

South Perth 6151. Western Australia

(Accepted 19 September 1989)

Summary In Western Australia, infection with cucumber mosaic virus (CMV) was

widespread in all three subspecies of subterranean clover (Trifolium subterraneum) growing in plots belonging to the Australian National Subterranean Clover Improvement Programme. Seed-borne CMV was detected in seed harvested in 1984-1986 of 18/25 cultivars from two collections of registered cultivars; seed transmission rates ranged up to 8.8%. Seed samples from CMV-inoculated plants of 11 cultivars transmitted the virus to 0.5-8.7% of seedlings. Seed transmission rates greater than 5% were obtained only with cvs Enfield, Green Range and Nangeela. CMV was not detected in seed harvested in 1975-1981 from one of the registered cultivar collections, in 17 commercial seed stocks from 1986 or in a survey of subterranean clover pastures.

Symptoms in subterranean clover naturally infected with CMV included mottle, leaflet downcurling and dwarfing but severity varied with cultivar and selection. CMV isolates from different sources varied in virulence when inoculated to subterranean clover; two (both from subterranean clover) were severe, two moderate and three (including one from subterranean clover) mild. In pot tests, CMV decreased herbage production and root growth (dry wts) of cv. Green Range by 49% and 59% respectively. In spaced-plants growing in plots, CMV decreased herbage production and root growth of cvs Green Range and Northam by 59-63070 and seed production of cv. Green Range by 45%. In rows sown with infected seed, aphid spread increased infection levels to 75% in cv. Green Range and 44% in cv. Esperance and losses in herbage production of 42% and 29% respectively were recorded.

CMV isolated from subterranean clover included isolates from both serogroups.

Key words: Virus, seed, pasture, clover, collection, losses, infection

Introduction Subterranean clover (Trifolium subterraneum) is well adapted to growing in self-regenerating

pastures in medium-high rainfall (> 350 mm annually) districts of the south west of Western Australia which has a predominance of sandy soils and a Mediterranean climate. Sowing of subterranean clover in the region began in the 1930s and increased dramatically in the 1950s and 1960s, the area sown reaching about 7 million hectares by the early 1970s (Gladstones, 1975; Gladstones & Collins, 1983; Collins & Gladstones, 1984; Collins, Francis & Quinlivan, 0 1990 Association of Applied Biologists

Page 2: Seed-borne cucumber mosaic virus infection of subterranean clover in Western Australia

74 R. A. C. JONES AND S. J. McKIRDY

1984). However, during the 1970s pasture deterioration problems were reported with increasing frequenc) , partly caused by diseases (Gillespie, 1983). Fungal and viral diseases can limit the productic n of subterranean clover by decreasing both the amount of foliage for fodder and the seed .Jield for future pasture regeneration (Barbetti, 1983; Barbetti & MacNish, 1983; McLean, 1983; Johnstone & Barbetti, 1987; Johnstone & McLean, 1987).

Cucumber mosaic virus (CMV) was first found infecting subterranean clover in 1983 at Denmark, Western Australia in a plot of cv. Enfield; symptoms were mild mottle, leaflet downcurlmg and dwarfing (McLean & Price, 1984). By 1986, the virus was widespread in subterranean clover breeding and evaluation plots in Western Australia. In 1987, seed-borne infection was detected and a major part of the subterranean clover seed increase programme was abandoned because of CMV infection (Jones, 1988a,b). Garrett (1987) reported widespread infection in subterranean clover plots in Victoria and showed CMV was seed-borne in the species. The virus has also been found infecting subterranean clover plants growing as weeds in CMV- nfected fields of narrow-leafed lupin (Lupinus angustifolius) in South Australia (Alberts, Hannay & Randles, 1985) and Western Australia (Jones, 1987, 1988~). This paper reports ar investigation of the occurrence and importance of seed-borne CMV in subterranean clover in Western Australia.

Materials and Methods Virus isolates and antisera. Seven isolates of CMV were used: LD and LW, from lupin,

and SN, ~‘rom subterranean clover, were from previous work (Jones, 1988~); SE was newly isolated l’rom subterranean clover cv. Esperance in Western Australia; SL was from Subterranean clover cv. Larisa in Victoria, supplied by R. G. Garrett; WC was newly isolated from white clover in Tasmania; and S was newly isolated from lesser snapdragon, Misopates orontium, in Western Australia. The isolate of alfalfa mosaic virus (AMV) used (EW) was from preirious work (Jones, 1988~). Cultures of both viruses were maintained in Nicotiana glutinosa or subterranean clover cv. Daliak. Antisera to CMV and AMV were supplied by R. I. B. Francki, Waite Agricultural Research Institute, Adelaide.

Manual Inoculations. Leaves were ground in 0.02 M phosphate buffer, PH 7.2, and the sap mixed with ‘Celite’ and rubbed onto leaves of subterranean clover or test plants. Leaf samples were tested for CMV infection by inoculation to Chenopodium amaranticolor or C. quinoa in which necrotic and/or chlorotic local lesions developed. Leaf samples were tested for bean yellow mosaic virus (BYMV) by inoculation to broad bean which developed a mosaic and C. quinotr in which necrotic or chlorotic local lesions developed.

Plants. Plants were grown in steam sterilised potting mix (mix of soil, sand and peat) in insect-proofed glasshouses. A moist peat inoculant (Nodulaid C ) of the root nodule bacterium Rhizobium leguminosarum var. triyolii was added to the potting mix for subterranean clover plants.

Produtivity trials. Seedlings grown from healthy seed of subterranean clover were transplanied into Jiffy pots and inoculated with isolate SN-infected N. glutinosa sap, mock- inoculated with sap from healthy N. glutinosa or left uninoculated. Jiffy pots were then transferred to 12.5 cm diameter plastic pots filled with sterilised potting mix and the pots kept in the glasshouse (pot trial) or transplanted into irrigated rows outside at South Perth 1-4 days after inoculation (spaced-plant trials). In the pot trial, pots were arranged in eight rows of alternately three healthy or three virus-inoculated pots. In the spaced-plant trials, transplani s were arranged in five pairs of rows 1 m in length (5 plants/row), one row of virus- inoculated and the other of healthy transplants. The rows were 1.5 m apart and barrier rows

I

Page 3: Seed-borne cucumber mosaic virus infection of subterranean clover in Western Australia

Seed-borne CMV infection of subterranean clover 15

of wheat (cv. Cranbrook or cv. Spear) were sown between them. The positions of the two treatments alternated from one pair to the next. Herbage and roots were collected separately from each plant 15 wk after infection and the roots thoroughly washed. Then both were dried in an oven at 60°C for three days and weighed. Burrs were harvested, 8 wk later, at the end of the growing season, threshed and the seeds cleaned and weighed.

In productivity trials with rows, healthy and CMV-carrying seed lots were sown alternately in paired irrigated rows at South Perth. The rows were 2 m apart and barrier rows of wheat cv. Cranbrook or oats cv. Swan were sown between them. Herbage collected near the end of the growing season was dried and weighed.

For statistical analysis (analysis of variance) of herbage and root dry weight data, pairs of adjacent virus inoculated and healthy plants (pot trial) or pairs of plants in corresponding positions in the paired virus-inoculated transplant and healthy transplant plots (spaced-plant trials) were treated as replicates. With seed weight data from spaced-plant trials and herbage data from row trials, pairs of plots containing virus-infected plants (spaced-plant trials) or which were partially infected (row trials) and healthy plants were treated as replicates and mean weight/plant or weight/lm of row values respectively obtained for each plot. These values were used for the analysis of variance.

Gel double diffusion tests. Leaves were extracted (1 g/4 ml) in 0.5 M sodium citrate, pH 6.8, containing 5 mM EDTA and 2 ml/litre thioglycollic acid (Francki & Hatta, 1980) and the extracts tested as described by Jones (1 988c).

Enzyme-linked immunosorbent assay (ELISA). Leaves were tested for CMV after extraction (1 g/20 ml) in 0.5 M sodium citrate, pH 6.8, containing 5 mM EDTA, 20 g/litre polyvinyl pyrrolidone, 1.5 ml/litre Tween 20 and 2 ml/litre thioglycollic acid (SCE). The extracts were tested by ELISA as described by Jones (1988~).

Leaves were tested for AMV by extracting (1 g/20 ml) in phosphate-buffered saline (10 mM potassium phosphate, 0.15 M sodium chloride), pH 7.4, containing 1.5 ml/litre Tween 20 and 20 g/litre polyvinyl pyrrolidone and testing by ELISA as described by Jones & Pathipanawat (1989).

Tests for seed transmission. Grouped samples of leaves from seedlings were either ground in a mortar and pestle with 0.2 M phosphate buffer, pH 7.2 (1 g/3 ml) and the extracts tested by manual inoculation to C. amaranticolor or C. quinoa, or ground, using a leaf press (Pollahne), in SCE and the extracts tested by ELISA. The groupings used varied from 5-50 and depended on the expected level of CMV infection determined by preliminary tests on large groups. Percentage seed transmission was estimated from grouped sample results as described by Jones (1982) using the formula of Gibbs & Gower (1960).

Electron microscopy. Samples prepared from diluted infective sap were stained with 2% neutral sodium phosphotungstate and examined in a Siemens Elmiskop 102 Electron microscope.

Results

Occurrence in plots In spring 1986, c. 20% of irrigated single row plots of subterranean clover cultivars and

breeders’ selections at South Perth and more than 50% of similar plots at the University of Western Australia field station at Shenton Park contained plants with mild mottle and leaflet downcurling (downcurling of leaflet laminae on either side of the midrib) sometimes associated with chlorosis and marked dwarfing (Fig. 1). When leaf samples from plants with these

Page 4: Seed-borne cucumber mosaic virus infection of subterranean clover in Western Australia

76 R. A. C. JONES AND S. J. McKIRDY

Fig. 1. I owncurling of leaflet laminae on either side of the midrib, mild mottle and chlorosis caused by CMV in a plot of subterranean clover cv. Esperance.

symptoms were tested by inoculation to C. amaranticolor and gel-diffusion serology, CMV was consis ently detected.

In 1987, subterranean clover plots at four sites (Medina Research Station, Many Peaks, Shenton Psrk and South Perth) were tested for CMV by ELISA. Infection was very widespread (Table 1) and symptoms were obvious in most of the affected plots. Selections belonging to subspecies abterraneum, yanninicum and brachycalycinum were all infected. At Shenton Park, where the initial phase of the Australian National Subterranean Clover Improvement Programm,: (NSCIP) is based, the virus was detected in some breeders' selections being grown as single spaced plants for use as parents in crossing. At Many Peaks, CMV was also detected by ELISA !n plots of shaftal clover (T. resupinatum) cv. Kyambro, white clover (T. repens) cv. Haifa m d balansa clover (T. balansa) cv. Paradana, but not in strawberry clover (T. fragiferum~ cv. Princep Park. When leaf samples from 50 of the plots tested at South Perth were also tested for AMV by ELISA, this virus was found in seven of them.

Table 1. Occurrence of cucumber mosaic virus in plots of subterranean clover*

Site Selections Named cultivars

Medina R.S. 28/50? - Many Peaks 9/10 5/6 Shenton Park 2W26 15/24 South Perth 19/98 9/25

* Samples (1 leafiplant) were taken from 25 randomly-selected plants in each plot and the combined sample tested for CMV by I LISA. All plots were sampled in late spring (October or November in 1987). f No. of plot: with CMV infectionho. of plots sampled.

CMV was also widespread in the 1987 trial plots of subterranean clover at Denmark and Mt Barker, and the final phase of the 1987 seed increase programme at Karridale had to be abandoned because of CMV. At Karridale, c.35% of plants of advanced breeding lines of ssp. subterrmeum showed symptoms while 61 ssp. yanninicum plots had up to 80% infection (Fig. 2.).

Page 5: Seed-borne cucumber mosaic virus infection of subterranean clover in Western Australia

Seed-borne CMV infection of subterranean clover 77

Fig. 2. Seed increase plot of an advanced selection of subterranean clover ssp. yunninicum partially infected with CMV. The patches of more vigorous taller plants are healthy and the stunted areas infected.

Occurrence in pastures In spring 1987, surveys were made to determine the incidence of CMV in subterranean clover

pastures in localities widely dispersed throughout the medium and high rainfall districts of the south west of Western Australia. Two pastures of cv. Seaton Park, two of cv. Green Range and three of cv. Karridale were systematically sampled (100-500 leaves taken at random) and the samples tested by ELISA. Nearly 250 other pastures were inspected for virus-like symptoms and samples from suspect plants tested by ELISA. The pastures examined included both ungrazed (used for seed increase) and grazed, and the cultivars represented were Daliak, Dalkeith, Dinninup, Esperance, Geraldton, Green Range, Junee, Karridale, Larisa, Meteora, Northam, Nungarin, Seaton Park and Trikkala. No CMV was found in any of the leaf samples. The only virus detected was BYMV which was widespread throughout the region. Its presence was confirmed by inoculation to C. quinoa and broad bean, and by electron microscopy. In spring 1988, BYMV infection was again widespread in subterranean clover pastures but tests by ELISA on samples sent in by advisers and inspectors failed to detect CMV.

Symptoms and spread Symptoms of CMV first became evident in subterranean clover plots in mid-August (late

winter), showing up as small patches of affected plants. The symptom-affected areas then expanded to form bands (across single row plots) or larger patches which were readily visible by late September (Fig. 2). This pattern of spread was consistent with spread of CMV by apterous aphids. Sometimes plots became fully infected before the end of the growing season in November. Severity of symptoms and extent of plot infection varied with selection and cultivar.

Two separate collections of the 25 currently registered cultivars of subterranean clover are propagated in plots at South Perth and Shenton Park each year. Plots of both collections were examined for CMV symptoms in 1986 and 1987 and presence of the virus in affected plots confirmed by tests. No symptomless infection was found. Symptoms were mild mottle, leaflet downcurling and dwarfing; chlorosis was a further symptom in some cultivars, particularly Esperance and Northam. Ssp. yanninicum cvs Meteora and Trikkala developed only mild reactions. Moderate reactions developed in ssp. yanninicum cv. Yarloop and ssp. subterraneum cvs Bacchus Marsh, Enfield, Green Range, Howard, Karridale and Nangeela. Severe or very severe reactions (Fig. 1) developed in ssp. brachycalycinurn C.V. Clare and subterraneum cvs Dalkeith, Daliak, Dinninup, Esperance, Junee, Northam, Nungarin,

Page 6: Seed-borne cucumber mosaic virus infection of subterranean clover in Western Australia

Tab

le 2

. Sym

ptom

s ca

used

by

six

cucu

mbe

r m

osai

c vi

rus

isol

ates

in s

ubte

rran

ean

clov

er c

ultiv

ars*

Cul

tivar

SN

w

c L

D

LW

SE

S

ssp.

sub

terr

aneu

m

Dal

keith

D

alia

k D

inni

nup

Espe

ranc

e G

reen

Ran

ge

June

e K

arri

dale

N

an g e

e I a

Nun

gari

n Se

aton

Par

k

ssp.

yan

nini

cum

Met

eora

M,S

Dc,

SDw

,Ct

M,S

Dc,

SDw

M

,SD

c,SD

w,C

M

,SD

c,SD

w,C

M

,SD

c,SD

w,C

M

,SD

c,SD

w,C

M

,SD

c,SD

w

SDc,

SDw

,C

M,S

Dc,

SDw

M

,SD

c,SD

w,C

M.D

c,D

w,C

M

,Dc,

Dw

M,D

c,D

w,C

M

,Dc,

Dw

,C

M,D

c,D

w,C

M

,Dc,

Dw

M

Dc,

Dw

,C

Dc,

Dw

-

-

M,M

Dc,

Dw

M

Dc,

MD

w

M,D

c,D

w,C

M

,Dc,

Dw

,Ld

M, D

c, D

w ,C

M

,Dc,

Dw

,C

Dc,

Dw

,P

M,D

c,D

w,C

M

,Dc,

Dw

,Ld

M,M

Dc,

MD

w

Dc,

Dw

-

M,M

Dw

,Ld

-

-

MD

c -

-

M,M

Dc,

MD

w,R

M

,MD

c,M

DW

,R

M,M

Dc,

MD

w,R

M

Dw

,R

MD

c,M

Dw

,R

MD

w

M,D

c,D

w,C

ss

M

,MD

c,M

Dw

-

MD

c,M

Dw

M

Dc,

MD

w

MD

c,M

Dw

M

Dc,

MD

w,C

M

Dc,

MD

w,C

M,M

dw,R

M

Dc

M,M

Dc,

MD

w,R

M

,MD

c,M

Dw

M

,MD

c,M

Dw

ss

M

,MD

c,M

Dw

M

Dc,

MD

w

MD

c,M

Dw

M

Dc,

MD

w,C

ss

ss

ss

* Pl

ants

wer

e m

anua

lly i

nocu

late

d w

ith in

fect

ed N

. gf

utin

osa

sap.

CM

V w

as d

etec

ted

in ti

p le

aves

of

inoc

ulat

ed p

lant

s by

ELI

SA.

73 ?

c, z x

t C

oded

sym

ptom

des

crip

tions

: M, m

ild m

ottle

or m

osai

c; M

Dc,

mild

leaf

let d

ownc

urlin

g; D

c, le

afle

t dow

ncur

ling;

SD

c, s

ever

e le

afle

t dow

ncur

ling;

MD

w, m

ild p

lant

dw

arfi

ng;

Dw

, pl

ant

dwar

fing

; SD

w, s

ever

e pl

ant

dwar

fing

; L

d, l

eaf

defo

rmat

ion;

C,

chlo

rosi

s or

lea

f pa

llor;

R,

reco

very

; S

S, s

ympt

omle

ss sy

stem

ic in

fect

ion;

-,

not

test

ed.

Page 7: Seed-borne cucumber mosaic virus infection of subterranean clover in Western Australia

Seed-borne CMV infection of subterranean clover 79

Fig. 3. Downcurling of leaflet laminae and severe dwarfing in subterranean clover cv. Daliak inoculated with CMV isolate SN (top); healthy plant (bottom).

Fig. 4. Downcurling of leaflet laminae and moderate dwarfing in plants of subterranean clover cv. Green Range inoculated with CMV isolate SN (left); healthy plants (right).

Seaton Park and Woogenellup. The virus was also found in ssp. subterraneum cvs Mt Barker and Tallarook but not in cvs Geraldton, Dwalganup, Larisa or Uniwager. By the end of the growing season infected single-row plots of most cultivars were still only partially affected, but those of cvs Enfield and Green Range were often fully affected. Fully affected plots were sometimes also found with cvs Esperance, Nangeela and Woogenellup. The eventual extent of plot infection apparently reflected the level of infection in the seed sown.

Symptoms caused by different virus isolates Six CMV isolates were inoculated to a range of subterranean clover cultivars and the

inoculated plants observed in the glasshouse for symptom development and tested for CMV by ELISA. As in naturally infected plants of the same cultivars, the predominant symptoms were mild mosaic, leaflet downcurling and plant dwarfing (Figs 3 and 4; Table 2), but chlorosis was more frequent under glasshouse conditions. Isolate LD induced leaf deformation as an additional symptom in three cultivars. In general, isolate SN induced the most severe reactions, isolates WC and LD caused moderate reactions while mild symptoms or symptomless infection resulted from infection with isolates LW, S and SE. The symptoms induced by isolates SN (from subterranean clover) and WC most closely resembled those seen in naturally infected plots. The second subterranean clover isolate (SE) gave mild symptoms except in cv. Junee, but the third (SL) caused severe symptoms in cvs Green Range and Junee, like those induced by isolate SN in these cultivars. In General, cvs Dalkeith, Nungarin and Seaton Park were very sensitive to CMV infection, cvs Daliak, Dinninup, Esperance and Junee were sensitive, cvs Enfield, Green Range, Karridale and Nangeela were of more moderate sensitivity, and cv. Meteora was the most tolerant.

Page 8: Seed-borne cucumber mosaic virus infection of subterranean clover in Western Australia

80 R. A. C. JONES AND S. J . McKlRDY

Table 3. Seed transmission of cucumber mosaic virus in 25 subterranean clover cultivars from two sites *

Cultivar

Bacchus Marsh Dalkeit h Daliak Dinninup Dwalganup En field Esperance Geraldton Green Range Howard Junee Karridale Nangeela Northam Mt Barker Nungarin Seaton Park Tallarook Uniwager Woogenellup

Larisa Meteora Tri k kala Yarloop

Clare

Shenton Park

No. of seedlings 070 infection tested of seedlings

ssp. subterraneum

125 0 150 0 200 0 200 1.9 120 0 100 8.8 I50 0.7 I75 0 152 7.7 125 0 200 1.9 200 2.7 150 6.9 175 0 140 4.2 200 0 I50 0.7 200 0.5 200 0 200 0.5

ssp. yanninirum

175 0.6 I40 0.8 I50 0 200 1.9

ssp. brachycalycinum

120 0.9

South Perth

No. of seedlings 070 infection tested of seedlings

80 0 325 0 240 0 275 0 140 0 135 7.8 I05 1 .o I20 0 60 4.0

100 0 292 0 I40 0 I10 0 160 0.6 200 0 105 1 .o I40 0 280 0 I40 0 70 0

100 0 75 1.4 75 1.4

205 0

I60 0.7

* Leaf samples from seedlings were grouped and tested by inoculation to C. amaranficolor or C. quinoa or by ELISA (grouping levels 5-25). Percentages were estimated using the formula of Gibbs & Gower (1960).

Relative virulence of the different CMV isolates on subterranean clover was not related to CMV serogroup (sensu Francki & Dietzgen). When tested at the Waite Institute, Adelaide, using monoclonal antibodies to CMV, SE and SL belonged to the serogroup containing French DTL, Japanese Y and American NY strains, while LW, LD, SN, WC and S belonged to the serogroup containing French TRS, Japanese P and American LC strains (R.I.B. Francki 8z R. Dietzgen, personal communication).

Seed transm ission Small seed samples from the South Perth and Shenton Park collections of registered cultivars

of subterranean clover were sown and the seedlings tested by inoculation to C. amaranticolor or C. quinoa, or by ELISA; all samples were from plots grown in 1984-1986. Seed-borne CMV

Page 9: Seed-borne cucumber mosaic virus infection of subterranean clover in Western Australia

Seed-borne CMV infection of subterranean clover 81

was detected in 18 cultivars belonging to all three subspecies (Table 3). Highest rates of seed transmission were obtained with cvs Enfield, Green Range, Nangeela, Karridale and Mt Barker all of which are late-maturing ssp. subterraneum types. No symptoms were seen in infected seedlings which were always tested when small and then discarded.

Because the extent of CMV infection in the original plots from which the infected seed lots in Table 3 were harvested was unknown, in 1987 all apparently healthy plants in partially infected plots of cvs Howard, Junee, Northam, Mt Barker, Meteora and Woogenellup were removed in late spring so that seed harvested after maturity all came from plants with symptoms. Seed was also harvested from fully infected plots of cvs Enfield, Esperance, Green Range and Nangeela and from plants of cvs Karridale and Junee infected with isolate SN in the glasshouse. In general, for each cultivar, the rates of seed transmission obtained were similar to those listed in Table 3 (Table 4).

In an attempt to find when CMV first contaminated the plots at Shenton Park, ten different seed stocks from 1975-1979 were tested for seed transmission by ELISA; the cultivars (numbers of seedlings of each tested in parentheses) were Daliak (134), Dinninup (140), Dwalganup (loo), Esperance (165), Geraldton (456) and Nungarin (175). No CMV was detected. Similarly no seed infection was detected in 14 seed stocks harvested in 1980-1981 of cultivars Bacchus Marsh (26), Dinninup (175), Esperance (247), Geraldton (225), Larisa (lo), Mt Barker (loo), Nangeela (23), Northam (loo), Nungarin (52), Seaton Park (loo), Tallarook (67) and

Table 4. Transmission of cucumber mosaic virus in seed from subterranean clover plants infected naturally or by manual inoculation *

No. of seedlings Cultivar (sample no) tested?

ssp. subterraneum

Enfield (1) Enfield (2) Esperance Green Range Howard Junee ( I ) Junee (2) Karridale Nangeela (1) Nangeela (2) Northam Mt Barker Woogenellup

270 ( 5 )

360 (20) 420 (10) 540 (20) 440 ( 1 0) 110 (10) 160 (10) 480 ( 1 0) 300 (15) 420 (20) 350 (25) 350 (25)

300 ( 5 )

ssp. yanninicum

Meteora (1) 220 (10) Meteora (2) 150 (10)

% infection of seedlings*

3.2 4.8 I .6 8.1 1 .o 1.5 4.4 2.1 3.1 3.9 1.3 4.0 2.2

0.5 0.7

* The Junee 2 and Karridale samples came from glasshouse grown plants manually inoculated with isolate S N , all other samples came from CMV-infected plants growing in 1987 in plots from which all apparently healthy plants had been removed.

t Leaf samples from seedlings were grouped and tested by ELISA. The figures in parentheses show thegroupings used.

rf Percentages estimated using the formula of Gibbs & Cower (1960)

Page 10: Seed-borne cucumber mosaic virus infection of subterranean clover in Western Australia

82 R. A. C. JONES AND S. J . McKIRDY

Fig. 5. Dwarfing in a row of subterranean clover cv. Green Range plants grown from transplants inoculated with CMV isolate SN (right); row grown from uninoculated transplants (left).

Woogenellup (90). Because of the low viability of old seed and the small size of remaining seed lots, testing of larger numbers of seedlings was not possible. These results suggest that CMV contamination was very low or absent prior to 1982.

Samples of commercial seed harvested in 1986 from 17 pastures in medium and high rainfall districts in the south-west of Western Australia (localities ranging from Eneabba in the north to Esperance and Karridale in the south) were sown and 500-2000 seedlings of each tested by inoculation to C. amaranticolor or C. guinoa, or by ELISA. No CMV was found suggesting that infection may not yet have spread from seed increase plots into commercial pastures. The cultivars tested were Dalkeith (6 sites), Green Range (2), Meteora (2), Nungarin (2) and Seaton Park (5).

Table 5. Effect of cucumber mosaic virus on productivity of subterranean clover transplants*

Expt

1

2

No. of Dry wts (g) Dry wts (9) Clean seed plants/ of herbage, of roots, WtS (PI,

Cultivar treatment Treatment meadplant meadplant meadplant

Pot experiments

Green Range 12 Healthy 29.35 14.24 -

CMV-infected 15.07 5.85 - S.E.D. (D.F.) 2.13 (11) 1.53 ( 1 1 ) -

Plot experiments

Northam 13 Healthy 64.44 -

CMV-infected 26.41 -

S.E.D. (D.F.) 9.26 (12) -

Green Range 19 Healthy 51.49 6.07 -

CMV-infected 18.91 2.34 - S.E.D. (D.F.) 6.07 (18) 0.72 (18) -

8.08 CMV-infected - - 4.41 S.E.D. (D.F.) -

Green Range 22 Healthy - -

- 0.63 (4)

* Young plants were manually inoculated with isolate SN, left uninoculated (cv. Green Range) or mock-inoculated with healthy sap (cv. Northam) and transplanted to pots (Expt 1) or in plots outside (Expts 2-4).

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Seed-borne CMV infection of subterranean clover 83

Effects on productivity In a pot experiment with cv. Green Range, all virus-inoculated plants became infected

(Fig. 4) and at 15 wk, herbage and root dry wts were decreased by 49% and 59% respectively by infection with CMV (Expt 1, Table 5 ) .

In spaced-plant experiments with cvs Green Range and Northam, most virus-inoculated plants developed symptoms (Fig. 5). Those without symptoms were removed along with the corresponding plant in the control row pair. Herbage and root dry wts were decreased by c. 6OVo and clean seed wt by 45% (Table 5). CMV spread from the infected rows to two of the control rows shortly before the end of the growing season in Expt 4 so the potential effect of CMV on seed yield may be underestimated.

In the row experiments, obvious CMV symptoms were evident prior to harvest in 70-80% of the row length in the cv. Green Range plots sown with infected seed, but in plots sown with infected cv. Esperance seed the extent of infection was more variable, ranging from 30-80%. No CMV symptoms were seen in plots sown with healthy seed. Overall plot infection levels of 75% for cv. Green Range and 44% for cv. Esperance resulted in losses in herbage dry wts of 42% and 29% respectively (Table 6).

Table 6. Effect of cucumber mosaic virus on herbage production in subterranean clover rows sown with infected seed*

Expt Cultivar

1 Green Range

2 Esperance

Vo CMV seed transmission Vo plants infected Dry wts (9) of herbage in seed sown at harvest mean11 m of row

0 9.4

0 1.6

0 1949 75 1127

S.E.D. (D.F.) = 162 (4)

0 803 44 57 1

S.E.D. (D.F.) = 38 (6)

* Single row plots 1 . 5 m (Expt 1) or 1.0 m long (Expt 2) were sown at seeding rates of 1 g/m (Expt 1) or 2 g/m (Expt 2). Herbage was harvested 19 wk (Expt 1 ) or 21 wk (Expt 2) after seeding and dry wts obtained; Expt 1 was done in 1987 and Expt 2 in 1988.

Discussion The high incidence of CMV found in subterranean clover plots shows that infection with

CMV is widespread in the NSCIP based in Western Australia. Further tests revealed frequent infection of seed stocks. Infected seed and subsequent spread to healthy plants by aphids both contributed to the extent of infection.

Apart from AMV which was not clearly associated with any symptom in naturally infected plants, the only other virus found infecting subterranean clover plots was BYMV which caused interveinal chlorosis, mottle, leaf deformation and a degree of dwarfing. BYMV infects a small proportion of plots every year often spreading into them late in the growing season. It was first reported in subterranean clover in Western Australia in plots in 1941 (Norris, 1943) and in pastures in 1954 (Harvey, 1956). It is now widespread and a probable cause of pasture deterioration. In addition to BYMV, another seed-borne virus, subterranean clover mottle virus, probably also causes pasture deterioration. Surveys indicate that it is widespread in pastures in the highest rainfall districts of Western Australia and often causes severe

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84 R. A. C. JONES AND S. J. McKIRDY

symptoms (Francki et al., 1983; Francki, Randles & Graddon, 1988; Graddon & McLean, 1988; K. Helms & P. M. Waterhouse, personal communication; J . M. Wroth & R. A. C. Jones, unpublished results).

Under glasshouse conditions, Garrett (1987) found that CMV caused a c. 60% loss of herbage production in cvs Enfield, Woogenellup and Yarloop. Similar losses were obtained in spaced-plant trials with cvs Green Range and Northam but a somewhat smaller loss in a glasshouse test with cv. Green Range. Smaller effects might be expected with cvs Meteora and Trikkala which are tolerant and greater effects with very sensitive cultivars (e.g. Dalkeith, Nungarin and Seaton Park). In a spaced-plant trial with cv. Green Range, CMV also decreased seed yield by 45%. These results and the results of the row trials presented here suggest that the virus has the potential to cause significant losses in subterranean clover pasture productivity and regeneration although such small scale trials need to be interpreted with caution as they do not reproduce the conditions found in sheep pastures where plants grow in dense swards and are grazed.

At present, CMV does not appear to be a significant cause of pasture deterioration in Western Australia. The authors failed to detect it in subterranean clover pastures except where trials or seed increase plots had been sown in them and did not find it in commercial seed stocks. This may be because the pastures were all sown with healthy seed. However, CMV may also persist poorly from year to year in annual legume pastures, perhaps as a result of poor establishment of infected clover seedlings in autumn, competition between infected and healthy plants and the effects of grazing.

Levels of CMV seed transmission found varied with cultivar and seed lot, but no CMV was detected in tests on pre-1982 seed stocks and if CMV was present in them it must have been at a very low level. The blue-green aphid, (Acyrthosiphon kondoi] which first arrived in Western Australia in 1979 (Sandow, 1980; Gillespie, 1983), is now the commonest aphid species in subterranean clover plots and pastures. Its arrival and rapid distribution coincided with the outbreak of CMV in the NSCIP. CMV was reported in the state prior to 1979, infecting passionfruit and a wide range of cucurbits, vegetables and ornamentals (McLean & Price, 1984). The virus may have originally spread into subterranean clover from such sources or may have been imported in seed introduced from the mediterranean region where subterranean clover originated (Gladstones & Collins, 1983). Infected seed lots have been distributed from the NSCIP in Western Australia to trial sites in five other Australian states and may be responsible for the outbreak of CMV in plots in Victoria reported by Garrett (1987).

Up until 1988, aphicides were applied to subterranean clover plots only when high populations of colonising aphids were noticed. Otherwise, no measures were taken which might help minimise spread of aphid-borne virus diseases. An ideal environment for cross infection of plots therefore existed. In 1988 at three sites (Shenton Park, South Perth and Medina), virus spread was decreased by regular application of aphicides, separating plots with barrier rows of oats and roguing out plants with symptoms. Weeding was also more thorough than previously as Jones (1987, 1988~) had found CMV in 15 species of weeds and volunteer legumes at the South Perth site. However, it was found that reflective mulch (Tachibana, 1981; McLean, Burt, Thomas & Sproul, 1982) was unsuitable for protecting single row plots of subterranean clover because its runners tended to cover the mulch, its burrs could not bury themselves and available soil water was restricted.

In 1988, a clean up programme was commenced for selected, advanced subterranean clover selections being propagated for seed increase and distribution within Australia and also for selected parental lines. Seedlings grown in Jiffy pots in the glasshouse were tested for CMV by ELISA and, if healthy, transplanted in an isolated site (Wooroloo) not previously used for subterranean clover. In spring all the plants were tested and any reinfected ones discarded. In 1989, the seed harvested will be propagated in plots at the same site and tested single plants

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Seed-borne CMV infection of subterranean clover 85

of further selected lines will be introduced. The eventual aim is to produce clean seed stocks for trialing, to ensure that new cultivars released are healthy or contain minimal levels of CMV and to clean up the collection of parental lines. To prevent introduction of CMV sources to new sites, sowing of healthy commercial seed stocks of established cultivars would also be necessary. Tests on CMV in seed stocks should be included in the seed certification regulations for newly released cultivars and for commercial seed stocks of established cultivars to help ensure this.

Garrett (1987) reported that plots of ssp. yanninicum cvs Meteora and Trikkala were tolerant of CMV while cv. Larisa apparently carried a degree of resistance to CMV infection by aphid vectors. Cvs Meteora and Trikkala were also found to be tolerant while cv. Larisa was one of four of the 25 currently registered subterranean clover cultivars in which infection was not detected in plots. However, CMV was detected in a seed sample of cv. Larisa. Use of cultivars or selections with resistance to CMV infection as parents and avoiding using parents giving high levels of seed transmission would seem to be advisable in breeding new cultivars. The highly susceptible cv. Nangeela which gave seed transmission rates up to 6.9% was a parent of cvs Green Range and Karridale both of which gave high seed transmission rates.

Acknowledgements In addition to those mentioned in the text, we thank M. J. Barbetti, W. J . Collins, D.

A. Nicholas, C. J . B. Sykes, P. Nichols and Department of Agriculture advisers and seed certification inspectors for supplies of seed and/or leaf samples, A. R. Baker, Kylie Clarke, Felicity Jones and D. Van den Bosch for technical assistance and K. Sivasithamparam for advice. Parts of the work were supported by the Australian Wool Corporation, the Commonwealth Wheat Research Council and the Western Australian Herbage Seed Trust Fund. S. J. McKirdy’s portion of the work formed part of a B.Agr.Sc. project with the Uhiversity of Western Australia.

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(Received 3 May 1989)

R. A. C. JONES AND S. J . McKlRDY