Control of Beet western yellows virus in Brassica napuscrops: infection resistance in Australian genotypes andeffectiveness of imidacloprid seed dressing

B. A. CouttsA,D, C. G. WebsterA,C, and R. A. C. JonesA,B

AAgricultural Research Western Australia, Department of Agriculture and Food, Locked Bag No. 4,Bentley Delivery Centre, Bentley, WA 6983, Australia.

BSchool of Plant Biology, Faculty of Natural and Agricultural Sciences, University of Western Australia,35 Stirling Highway, Crawley, WA 6009, Australia.

CCurrent address: United States Department of Agriculture-Agricultural Research Services (USDA-ARS),Fort Pierce, FL 34945, USA.

DCorresponding author. Email: brenda.coutts@agric.wa.gov.au

Abstract. Eighteen Brassica napus (canola) genotypes were examined for their responses to infection with Beet westernyellows virus (BWYV) and infestation by Myzus persicae (green peach aphid) in a field experiment and in a series of potexperiments under controlled-environment conditions. When exposed to infection with BWYV in the field, plants of cvv.Tranby and Trigold remained uninfected with BWYV. Only 2–5% of plants of cvv. Stubby and Banjo became infected,but infection incidence was 14–23% in cvv. Tanami and Jade, and reached 45–65% in 12 other commercial cultivars oradvanced breeding lines of B. napus. Once plants became infected, the sensitivity rankings for most genotypes were2–3: mild to moderate symptoms consisting of plant stunting and reddening of lower leaves. When plants of cvv. Tranby,Trigold, Stubby, and susceptible control cv. Pinnacle growing in pots were exposed to spread of BWYV by viruliferouswinged M. persicae flying from an infested cv. Pinnacle plant infected with BWYV, similar numbers of aphids colonisedeach of the different cultivars. Thus, no aphid feeding preference was apparent among the different B. napus cultivars.However, all 18 plants of cv. Pinnacle became infected with BWYV, but only 1, 2, and 5 plants of cvv. Trigold, Tranby,and Stubby became infected, respectively. When 68 plants each of cvv. Tranby, Trigold, and Stubby were each inoculatedwith 1–10 viruliferous aphids/plant, only 1 of cv. Trigold, 3 of cv. Tranby, and 6 of cv. Stubby became infected withBWYV despite infection of 45 plants of cv. Pinnacle. This shows that cvv. Tranby, Trigold, and Stubby have resistance toinfection with BWYV by aphid transmission. In 2 experiments when viruliferous M. persicae were placed on plants ofB. napus grown from seed treated with imidacloprid (240 g a.i./100 kg seed), they infested 72% of plants grown fromtreated seed and transmitted BWYV to 62% of them regardless of the growth stage inoculated. Aphids colonised 100% ofplants grown from untreated seed but 0% of plants sprayed with imidacloprid (2 g a.i./L water), and infection with BWYVwas diminished markedly by the foliar spray. This suggests that insufficient insecticide adhered to most of the dressedseeds to kill the aphids and prevent BWYV transmission. B. napus cultivars found to have infection resistance to BWYVcan be used in conjunction with imidacloprid seed dressings (if applied effectively) as components of an integrated diseasemanagement strategy for control of BWYV in B. napus crops.

Additional keywords: BWYV, Myzus persicae, virus, aphids, canola, oilseed rape, insecticide, virus resistance.

Introduction

Brassica napus (canola, oilseed rape) is grown widely aroundthe world as an oilseed crop, its oil being used for cookingand margarine, and its meal for animal feed (Raymer 2002).B. napus cultivars that produce seed with oil with erucic acidconcentrations of <2% and meal with <40mmol glucosinolates/gramare referred to as canola (Salisbury et al. 1999).B.napuswasfirst grown commercially in Australia in 1969. In the 1990sproduction increased dramatically with the development ofcultivars with improved oil quality and low glucosinolatelevels, resistance to Leptosphaeria maculans (cause of

blackleg disease), herbicide tolerance and improved yield. In2007 in Australia, the production of B. napus seeds was1.069million tonnes from a total area planted of 758 000 ha(Anon. 2008). In 2007, in the south-west Australian ‘grainbelt’the area sown toB. napuswas 390 000 ha. This ‘grainbelt’ regionhas a Meditteranean-type climate and B. napus crops are sownin late autumn to early winter after the first rains and harvested5–6 months later in late spring to early summer.

Beet western yellows virus (BWYV; family Luteoviridae,genus Polerovirus) is an important pathogen of B. napus andother Brassica species worldwide. The virus is phloem-limited

� CSIRO 2010 10.1071/CP09264 1836-0947/10/040321

CSIRO PUBLISHING

www.publish.csiro.au/journals/cp Crop & Pasture Science, 2010, 61, 321–330

and spread persistently by more than 10 different aphid species,with Myzus persicae (green peach aphid) the principal naturalvector (Duffus 1972; Brunt et al. 1996; Smith and Barker 1999).The viruses formerly known as BWYV have recently beenreclassified into 4 distinct virus species: BWYV, Turnipyellows virus (TuYV), Beet chlorosis virus and Beet mildyellowing virus all of which are closely serologically related.BWYV isolates shown not to infect sugar beet (Beta vulgaris)were reclassified as a separate species in the genus Polerovirusunder the name TuYV (Duffus and Russell 1972; Graichen andRabenstein 1996; Hauser et al. 2000, 2002; D’Arcy and Domier2005; Beuve et al. 2008). As mentioned previously (Coutts et al.2006; Jones et al. 2007), the sequence of BWYV isolate WA-1from south-west Australia resembles that of TuYV isolates fromEurope. However, insufficient Australian isolates have beensequenced to establish whether BWYV or TuYV predominate,so, as in our previous studies (Coutts and Jones 2000;Coutts et al.2006; Jones et al. 2007; Maling et al. 2010), the name BWYV isused here. Widespread BWYV infection of B. napus crops isimportant because it decreases seed yield and oil content andincreases erucic acid and glucosinolate contents significantly(Smith and Hinckes 1985; Schroder 1994; Graichen andSchliephake 1999; Jay et al. 1999; Jones et al. 2007). In the‘grainbelt’, BWYV has substantial yield limiting potential inB. napus crops and diminishes oil but increases the erucic acidcontents of seeds. In field experiments when BWYV incidencereached 96–100%, seed yield decreased by up to 46%, and forevery 1% increase in virus incidence there was a 6–12 kg/hadecline in yield (Jones et al. 2007).Moreover,BWYVinfection iswidespread in B. napus crops and Raphanus raphanistrum (wildradish) weeds (Coutts and Jones 2000). Brevicoryne brassicae(cabbage aphid), Lipaphis erysimi (turnip aphid) andM. persicaeare the only aphid species commonly found infesting B. napuscrops in the ‘grainbelt’. The first two species cause direct damageby feeding on the growing shoot tips causing wilting, flowerabortion and reduced pod set (Berlandier 2004), whileM. persicae also feeds on lower leaves and can kill or severelystunt young seedlings in the field (Jones et al. 2007).M. persicaeis the most important vector of BWYV, but B. brassicae can alsotransmit it at much lower efficiency (Schliephake et al. 2000).Berlandier and Valentine (2003) indicated that some AustralianB. napus cultivars may be more susceptible than others tocolonisation by B. brassicae and L. erysimi. However, thereare apparently no data on whether B. napus cultivars vary intheir responses to infestation with M. persicae.

In the ‘grainbelt’, 2 early foliar applications of pyrethroidinsecticide give effective control of Barley yellow dwarf virus(BYDV; family Luteoviridae, genus Luteovirus) in autumn sowncereal crops and provide a key component of the IntegratedDisease Management (IDM) approach used against thisluteovirus (McKirdy and Jones 1996, 1997; Thackray et al.2005, 2009). However, when 2 early foliar applications of thesame pyrethroid were applied to B. napus plots at similar rates, itwas ineffective in controlling M. persicae or spread of BWYV(Jones et al. 2007). The explanation is that in the ‘grainbelt’prophylactic use of insecticides such as organophosphates,carbamates and pyrethroids to control insect pests includingaphids and virus spread has led to insecticide resistance inaphids, especially M. persicae. However, so far, resistance to

neonicotinoid insecticides, such as imidacloprid, has not beenfound (Thackray et al. 2000; Edwards et al. 2008). Seed dressingwith the neonicotinoid insecticide imidacloprid is effective insuppressing spread of BYDV in cereals (McKirdy and Jones1996). Imidacloprid seed dressing used alone and applied bymixing in a glass jar at 525 g a.i./100 kg of seed effectivelycontrolled insecticide-resistant M. persicae and suppressedspread of BWYV in B. napus for 2.5 months, increasing seedyieldby84–88%(Jones et al. 2007).However, in2003–05, 6fieldexperiments (2/year) with this seed dressing applied at therecommended rate (240 g a.i./100 kg of seed) by commercialprocedures gave disappointing levels of control of M. persicaeand BWYV (Coutts and Jones 2005; Jones et al. 2007).

In the USA, Thomas et al. (1990) evaluated 4 forage cultivarsof B. napus for resistance to BWYV by field exposure andreported that one had partial resistance to infection while theothers varied in relative susceptibility. In Germany, resistance toTuYV was identified in single plant selections from‘resynthesised’ B. napus breeding line R54. When Graichenand Peterka (1999) crossed R54 with B. napus cultivars, theprogeny plants segregated for TuYV resistance. To the best of ourknowledge, there has beennoother research onBWYVresistancein B. napus.

This paper reports the evaluation of 18 Australian B. napusgenotypes for their reactions to BWYV when exposed toinfection in the field. It also reports pot experiments doneunder controlled environment conditions that (i) evaluatedthree B. napus cultivars with potential infection resistance toBWYV for their responses toM. persicae feeding preference andcolonization, and to aphid-inoculation with the virus, and(ii) investigated why imidacloprid seed dressing applied at therecommended rate using commercial procedures is relativelyineffective at controlling M. persicae colonisation andpreventing BWYV-infection in B. napus.

Materials and methodsVirus isolate, plants, inoculations and antiserum

Test, culture and ‘infector’ plants were grown in insect-proof,air-conditioned glasshouses and maintained at c. 208C. Plants ofB. napus were grown in steam-sterilised potting mix containingsoil, sand and peat (1 : 1 : 1). For aphid inoculations,M. persicaewere starved for 2 h, and then placed on BWYV-infected leavesfor 2 days before feeding on healthy plants for 24 h before beingkilled with aphicide. BWYV isolate WA-1 (Coutts and Jones2000) was maintained by aphid inoculation to plants of B. napuscv. Pinnacle. Samples from this culture of BWYV were used asthe positive control in serological tests. Tissue blot immunoassay(TBIA) was used to test the petioles of leaf samples from (i) thefield experiment, and (ii) the pot experiments with insecticidalseed dressings, but enzyme-linked immunosorbent assay(ELISA) to test leaf samples from the pot experimentsexamining aphid feeding preference and inoculation. ‘Infector’plants of B. napus were produced by placing viruliferousM. persicae from colonies growing on B. napus plants infectedwith BWYV isolate WA-1 onto B. napus cv. Grace seedlings(5 aphids/plant) growing in peat pots, and leaving them in place tocolonise. B. napus cv. Pinnacle was included as a susceptiblecontrol in pot experiments as it was found to be susceptible in

322 Crop & Pasture Science B. A. Coutts et al.

previous work (Jones et al. 2007). Polyclonal antiserum toBWYV was obtained from Sediag-Diagnostics, France.

Tissue blot immunoassay

B. napus petioles were tested singly or petioles were bound inbundles of up to 10 with Parafilm. A scalpel was used to cut theends off single petiole or petiole bundle samples. The cut surfaceswere pressed twice onto 0.45mm pore size nitrocellulosemembrane (Schleicher and Schuell, Inc., Keene, NH, USA).The procedure for TBIA was as described by Coutts and Jones(2000).

Enzyme-linked immunosorbent assay

B. napus leaf samples were extracted singly or in groups of2–10 leaves in phosphate buffered saline (10mM potassiumphosphate, 150mM sodium chloride), pH 7.4, containing5mL/L of Tween 20 and 20 g/L of polyvinyl pyrrolidone(1 g leaf/20mL) using a mixer miller (Retsch, Germany). Theextracts were tested by double antibody sandwich ELISA usingpaired wells in immunoplates as described by Clark and Adams(1977). The substrate used was 0.6mg/mL of p-nitrophenylphosphate in 100mL/L of diethanolamine, pH 9.8.Absorbance values (A405 nm) were measured in a Microplatereader (Bio-Rad, Japan). Absorbance values more than tentimes those recorded for healthy leaf sap were considered torepresent infected plants.

Field exposure experiment

In July 2006, a field experiment which evaluated 18 B. napusgenotypes (14 commercial cultivars and 4 advanced breedinglines) for their responses to infection with BWYV was sown atMedina Research Station (328140S, 1158480E) (Table 1). Prior tosowing all seeds were treated with fungicide fluquinconazole(Jockey®) to control infection with L. maculans and a weed-freeseed bed was prepared. Single row plots 6m in length with3 replicates were sown in a randomised block design. Thedistances between the ends and sides of each plot were 1mand 2m, respectively. BWYV ‘infector’ plants infested withM. persicae were transplanted at either end of each test row(‘infector’ plants) and healthy B. napus plants transplanted inbetween test rows using the experimental design ofMcKirdy andJones (1995). This design ensured a high and uniform initialBWYV inoculum source for natural spread by aphids and build-up of the inoculum source subsequently as the healthy transplantsbecame infected by winged aphids. Single row plots werethinned to 60 plants each once all had germinated, and theplots were irrigated. At 69 and 98 days after sowing (DAS),a petiole sample was taken from every plant within each testrowand tested byTBIA forBWYV.At 112DAS, all plants in anytest rows in which no BWYVwas found in the previous sampleswere resampled and tested in the same way. Data for percentageof plants in which BWYV was detected were recorded foreach plot.

Susceptibility and sensitivity rankings

At 112 DAS, for each genotype the different types of viralsymptoms caused were recorded, and susceptibility andsensitivity rankings assigned. The different susceptibilityrankings used relate to the relative ease with which plants of a

given genotype became infected when exposed to virusinfection, while different sensitivity (= symptom severity)rankings refer to the intensity of the reactions in plants afterthey become infected. Susceptibility rankings were based onthe percentage of plants that became infected. There were5 categories ranging from HS, highly susceptible to HR,highly resistant. Plant sensitivity rankings were on a 1–5 scale:1, symptomless infection, to 5, extremely severe symptoms(McKirdy et al. 2000).

Pot experiments

All pot experiments were done in controlled environmentcabinets maintained at c. 208C with 12 h day/night length and180 micro Einsteins light intensity.

Aphid feeding preference and inoculation experiments

In Expt 1a, 9 plants at the 2 true-leaf stage of each of3 potentially BWYV-resistant B. napus cultivars (Stubby,Tranby and Trigold) and susceptible cv. Pinnacle werearranged in 4 groups around a central BWYV-infected,M. persicae-infested ‘source’ plant of B. napus of cv.Pinnacle. Within each group, the plants were arranged so thateach cultivar was represented in equivalent positions in alldirections and distances from the ‘source’ plant, and plantswere not touching each other (Fig. 1). Pots were placed on awire grill such that aphids could not walk from pot-to-pot.The number of aphids (winged, non-winged and nymphs)present on each plant was counted 7 days after their

Table 1. Australian Brassica napus genotypes evaluatedAgVic, Victoria Department of Primary Industries, Horsham, Victoria; CA,Canola Alliance, NSW Department of Primary Industries and Nugrain;CBWA, Canola breeders Western Australia, Perth, WA; DAFWA,Department of Agriculture and Food, Western Australia; Pacific, Pacific

Seeds, Toowoomba, Queensland

Genotype Yearreleased

Breeder Parentage

ATR Banjo 2006 AgVic 97-018T/97-103HATR Beacon 2002 AgVic Pinnacle selectionCB Boomer 2005 CBWA Karoo/Surpass 400Bravo TT 2005 CA TN1/BLN2055AV Jade 2005 AgVic RM30/RM17Rocket CL 2005 Pacific C9128/Surpass 603CLATR Stubby 2004 Monsanto 97-018T/97-100HCB Tanami 2006 CBWA Composite of 5 linesThunder TT 2005 Pacific Surpass 600TT/9102Tranby 2004 DAFWA 82N160-63A/586NO34-8//

KEM165/3/MontyCB Trigold 2004 CBWA Karoo/RL31-97M1CB Trilogy 2004 CBWA Karoo/Surpass 400 selectionTornado TT 2004 Pacific Surpass 600TT/9102Warrior CL 2005 CA 45A71/BLN1240TR006-03W30 – DAFWA Unknown advanced

breeding lineTR006-03W08 – DAFWA Unknown advanced

breeding lineTR002-03M09 – DAFWA Unknown advanced

breeding lineTR004-03M02 – DAFWA Unknown advanced

breeding line

Control of Beet western yellows virus in Brassica napus Crop & Pasture Science 323

introduction (DAI) into the cabinet and exposure to the ‘source’plant and then every 2 to 4 days up until 34 DAI when all plantswere sprayed with the carbamate aphicide pirimicarb (Pirimor®).When the plants were inspected daily afterwards, no aphidshad survived. One tip leaf from each plant was sampled andtested individually by ELISA for BWYV at 44, 58 and 72 DAI.Experiment 1bwas a repeat of Expt 1a, except that all plants weresprayed with aphicide at 25 DAI and a single tip leaf from eachplant was tested individually by ELISA for BWYV at 37, 49 and79 DAI.

Aphid inoculation experiments

In Expt 2a, plants at the 2 true-leaf stage of each of B. napuscvv. Pinnacle, Stubby, Tranby and Trigoldwere aphid inoculatedby placing 1, 3 or 6 viruliferousM.persicae upon them (10 plants/number of aphids/cultivar). A further 2 plants of each cultivarwere left uninoculated as a control. All plants were sprayed withaphicide (pirimicarb) 24 h after inoculation. When the plantswere inspected daily afterwards, no aphids were present.A single tip leaf sample was taken from each plant and testedat 28, 43 and 58 DAI. Experiment 2b was a repeat of Expt 2a,except that there were 2 inoculation treatments which consistedof either 1 or 2 viruliferous M. persicae/plant, and the sampleswere tested for BWYV at 30 and 45 DAI. Experiment 2c wasa repeat of Expt 2a, except that there was one inoculationtreatment in which 8 plants of each cultivar were inoculatedwith 10 viruliferous M. persicae, and the number of plants ofeach cultivar left as uninoculated controls was 8. All plants

were sprayed with pirimicarb 24 h after inoculation. A singletip leaf from each plant was sampled and tested for BWYV at24 and 41 DAI. Experiment 2d was a repeat of Expt 2a, exceptthat there was only one inoculation treatment in which 6viruliferous M. persicae were placed on each plant and eachplant was sampled and tested at 28 and 42 DAI. A further2 plants of each cultivar were left uninoculated as a control.

Insecticidal seed dressing experiments

For Expt 3a–d, the four treatments were plants of B. napuscv. Grace with and without foliar insecticide spray grownfrom seed previously dressed with imidacloprid (Gaucho®) at240 g a.i./100 kg using commercial procedures, and plants of cv.Grace with and without foliar insecticide spray grown from seedwithout any insecticide dressing. Ten plants were used for eachtreatment. For aphid inoculations, 5 viruliferous M. persicaewere placed on each plant. The foliar insecticide spray usedwas imidacloprid (Confidor 200SC® at 2 g a.i./L water) applied12 h after the aphids had been placed on the plants. ForExpt 3a–d, aphids were placed on B. napus plants at 18, 15(2–3 leaf stage), 32 and 29 (6–7 leaf stage) DAS, respectively.Aphids (non-winged and nymphs) were counted on each plantevery 2 to 4 days for a total of 8 to 29 DAI. A single leaf petiolefrom each plant was sampled and tested by TBIA at 14 to29 DAI.

Experiment 4a and 4b were repeats of Expt 3 except thatthe cultivar used was Beacon and the insecticidal seed dressingimidacloprid (Gaucho® at 240 g a.i./100 kg seed) was applied

BWYV Canola

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Fig. 1. Positioning of plants of four Brassica napus cultivars around a centrally positioned B. napus cv.Pinnacle ‘source’ plant infected with Beet western yellow virus and infested with Myzus persicae inExpt 1 a and 1b. Cultivar abbreviations used are: Pi, Pinnacle; St, Stubby; Tb, Tranby; Tg, Trigold. Eachexperiment was undertaken inside a controlled environment cabinet maintained at 208C, 12 h day/nightlength and 180 micro Einsteins light intensity. Distance of 10 cm was used between pots within eachgroup and between groups.

324 Crop & Pasture Science B. A. Coutts et al.

using a small-scale chemical seed applicator. Aphid inoculationwas done at 17 DAS (2–3 leaf stage) (Expt 4a) or 31 DAS (6–7leaf stage) (Expt 4b). The number of aphids (non-winged andnymphs) present on each plant was counted at 2, 7, 10, 13 and16 DAI (Expt 4a), or 1, 5 and 8 DAI (Expt 4b). A single leafpetiole from each plant was sampled and tested by TBIA at33 DAI (Expt 4a) or 14 DAI (Expt 4b).

Statistical analysis

Percentage BWYV infection data from the field experimentwere angular transformed before being subjected to analysis ofvariance (ANOVA) using GENSTAT version 8.

Results

Field exposure experiment

At 69 DAS, few rows were infected, the maximum BWYVincidence found being 2%. By 98 DAS, many rows hadbecome infected, with infection incidences for the differentgenotypes ranging from 0 to 63% (Table 2). The genotypestested ranged in susceptibility from highly resistant tosusceptible, but with no plants in the highly susceptiblecategory. At this time, cvv. Tranby and Trigold were ranked ashighly resistant. Cultivars Stubby and Banjo ranked as resistant(2–5% incidence) and cvv. Tanami and Jade as moderatelyresistant (14–23% incidence). The other 12 genotypes testedwere ranked as susceptible (45–63% incidences). At 112 DAScvv. Tranby and Trigold plants were resampled and tested

again but both remained without infection. The B. napusgenotypes that became infected ranged in sensitivity from 1to 4 (Table 2). There was no relationship between thesusceptibility and sensitivity rankings of genotypes, as thoseranked moderately resistant or susceptible had sensitivityrankings ranging from 2–4. No symptoms were seen in thefew BWYV-infected plants of cvv. Stubby or Banjo, whileinfected plants of cvv. Tanami, Thunder, Tornado and TR006-03W08 had verymild symptoms (2 ranking). All other genotypeshad moderate symptoms (3 ranking), except for cv. Rocket inwhich the symptoms were severe (4 ranking). The predominantsymptom types found were plant stunting and reddening of olderleaves.

Pot experiments

Aphid feeding preference and inoculation experiments

InExpt 1a and1b, colonising aphids (nymphs andnon-wingedadults) were first observed on plants of each cultivar at 16 and 7DAI, respectively (Table 3) but no winged aphids were seen onany plant in either experiment. In Expt 1a, by 27 DAI aphids hadcolonised all 9 plants each of cvv. Trigold and Stubbywith 10 and15 aphids/plant, respectively. All plants of cv. Pinnacle wereinfested by 30 DAI (21 aphids/plant), while all plants of cv.Tranby were infested by 34 DAI (17 aphids/plant). In Expt 1b,by 21 DAI all plants of cv. Trigold were colonised (15 aphids/plant). By 23 DAI, all plants of cvv. Tranby and Stubby wereinfested with 25 and 27 aphids/plant, respectively. For cv.

Table 2. Susceptibility and sensitivity of Brassica napus (canola) genotypes to field infection with Beet western yellows virusPercentage infection was determined by testing petiole samples 98 days after sowing from all plants from each test row for BWYV byTBIA. All percentage BWYV data were angular transformed before analysis. Back transformed percentage infection data are in bold.Genotypes with the same letter are not significantly different at P< 0.05. Coded susceptibility rankings: HR, highly resistant; R, resistant;MR, moderately resistant; S, susceptible. Coded sensitivity ranking scale 1, symptomless infection, to 5, severe symptoms. Codedsymptom descriptions: SI, symptomless infection; LR, lower leaf reddening;MLR,mild lower leaf reddening; S, plant stunting;MS,mild

plant stunting; NI, not infected

Genotype % infection Significance(from %infection)

Susceptibilityranking

Sensitivityranking

Predominantsymptoms

Tranby 1.8 0 a HR – NITrigold 1.8 0 a HR – NIStubby 9.0 2 ab R 1 SIBanjo 12.8 5 ab R 1 SITanami 21.6 14 bc MR 2 MLR, SJade 28.9 23 cd MR 3 LR, SBravo 42.4 45 de S 3 LR, STR006-03W30 44.6 49 de S 3 LR, STR006-03W08 44.8 50 de S 2 MLR, SBoomer 46.5 53 e S 3 LR, SBeacon 47.9 55 e S 3 LR, SWarrior 48.8 57 e S 3 LR, STornado 50.6 60 e S 2 MLR, MSTR002-03M09 50.9 60 e S 3 LR, STR004-03M02 51.5 61 e S 3 LR, SThunder 51.6 61 e S 2 MLR, MSRocket 52.1 62 e S 4 LR, STrilogy 52.5 63 e S 3 LR, S

P <0.001l.s.d. 16.11d.f. 34

Control of Beet western yellows virus in Brassica napus Crop & Pasture Science 325

Pinnacle, all plants were colonised by 25 DAI (28 aphids/plant).At final assessment (Expt 1a, 34 DAI; Expt 1b, 25 DAI), thenumber of aphids colonising each plant ranged from 17 (cv.Tranby, Expt 1a) to 34 (cv. Stubby, Expt 1a). In Expt 1a, amongthe 9 plants each of cvv. Trigold, Tranby andStubby, BWYVwasdetected in tip leaves from only 1, 2 and 3 plants, respectively. InExpt 1b, 2 plants of cv. StubbybecameBWYV-infected,while novirus was detected in tip leaves of any plants of cvv. Tranby orTrigold. However, BWYV was detected in tip leaves of all 9plants of cv. Pinnacle in both of Expt 1a and 1b.

Aphid inoculation experiments

For Expt 2a–d, regardless of the number of viruliferousaphids (1–10/plant) used to inoculate plants of cv. Trigold,only 1 plant ever became infected with BWYV, which waswhen 10 viruliferous aphids were used/plant (Expt 2c)(Table 4). With cv. Tranby, on one occasion 3 of 10 plantsbecame infected following inoculation with 6 viruliferousaphids/plant (Expt 2a), but, otherwise no plants ever becameinfected (Expt 2a–d). Plants of cv. Stubby became infectedwhen inoculated with 2 aphids/plant (3/10 plants) (Expt 2b),3 aphids/plant (2/10 plants) (Expt 2a), and 6 aphids/plant(1/10 plants) (Expt 2d). In contrast, with cv. Pinnacle someplants always become infected after inoculation withviruliferous aphids, ranging from 1 (Expt 2a, 1 aphid/plant) to

10 (Expt 2d, 6 aphids/plant) out of 10 plants. BWYV wasnever detected in any of the uninoculated control plants inExpt 2a–d.

Insecticidal seed dressing experiments

In Expt 3a–d, no aphids survived when foliar insecticide wasapplied after aphid inoculation to plants of cv. Grace regardlessof whether the seed sown was dressed or without insecticidaldressing, but 1 to 5 of 10 plants/experiment still became infectedwith BWYV (Table 5). In contrast, aphids colonised all plants ofcv. Grace grown from undressed seeds and left without foliarapplication, and at 3–5 DAI there were 5–10 aphids/plant; 4 to 10of 10 plants/experiment became infected with BWYV. Whenplants grown fromdressed seed and left without foliar applicationhad aphids placed on them at 18 or 15DAS (Expt 3a and 3b), 4–6of 10 plants were colonized at 4–5 DAI with a mean of 3 aphids/plant.When aphidswere placed at 32 or 29DAS (Expt 3c and 3d)on plants grown from dressed seed and left without foliarinsecticide application, 8–9 of 10 plants were colonized at 4–5DAI with means of 3–6 aphids/plant. When the plants grownfrom dressed seed and left without foliar application were testedfor infection with BWYV, 7–10 of 10 plants were found infected(Expts 3a–d).

In Expt 4a and 4b, when plants of cv. Beacon grown fromdressed seed and left without foliar insecticide application had

Table 3. Number of plants of four Brassica napus cultivars colonised with Beet western yellows virus-infective Myzus persicaein aphid feeding preference experiments (Expt 1a and 1b)

Aphid counts include non-winged adults and nymphs. The mean number of aphids colonising each plant is shown in parentheses. Foreach experiment, therewere 9 plants/cultivar arrangeduniformly around a centralBWYV-infected,M.persicae-infestedB. napus ‘source’plant (Fig. 1). Tip leaf samples from all plants were tested individually for BWYV by ELISA at 72 (Expt 1a) and 79 (Expt 1b) days after

introduction to the controlled environment cabinet containing the ‘source’ plant

Cultivar Days after plants introduced to ‘source’ plant No. of BWYV-infected plants

Expt 1a7 10 13 16 20 23 27 30 34

Pinnacle 0 0 0 2 (0.3) 4 (4) 4 (8) 7 (14) 9 (21) 9 (28) 9Stubby 0 0 1 (0.1) 4 (1) 6 (6) 8 (11) 9 (15) 9 (25) 9 (34) 3Tranby 0 0 2 (0.3) 3 (1) 6 (6) 6 (7) 7 (9) 8 (12) 9 (17) 2Trigold 0 0 0 2 (1) 7 (2) 7 (6) 9 (10) 9 (17) 9 (33) 1

Expt 1b7 9 11 14 16 18 21 23 25

Pinnacle 3 (0.5) 3 (1) 3 (2) 3 (4) 5 (9) 5 (12) 7 (17) 7 (20) 9 (28) 9Stubby 2 (0.2) 5 (2) 6 (3) 6 (7) 6 (14) 6 (18) 8 (23) 9 (27) 9 (32) 2Tranby 1 (0.1) 1 (0.4) 1 (0.4) 4 (3) 6 (6) 7 (10) 7 (19) 9 (25) 9 (28) 0Trigold 1 (0.1) 1 (0.6) 2 (1) 3 (2) 4 (5) 8 (10) 9 (15) 9 (24) 9 (32) 0

Table 4. Effect of number of viruliferousMyzus persicae used to inoculate plants on transmission of Beet western yellows virus to four Brassica napuscultivars (Expt 2a–d)

Tip leaf samples from each plant were tested individually for BWYV by ELISA at 58, 45, 41 and 42 days after aphid inoculation in Expt 2a–d, respectively.Figures are number of BWYV-infected plants/total number of plants inoculated

Cultivar Number of viruliferous aphids/plantExpt 2a Expt 2b Expt 2c Expt 2d

0 1 3 6 0 1 2 0 10 0 6

Pinnacle 0/2 1/10 4/10 9/10 0/2 7/10 7/10 0/8 7/8 0/2 10/10Stubby 0/2 0/10 2/10 0/10 0/2 0/10 3/10 0/8 0/8 0/2 1/10Tranby 0/2 0/10 0/10 3/10 0/2 0/10 0/10 0/8 0/8 0/2 0/10Trigold 0/2 0/10 0/10 0/10 0/2 0/10 0/10 0/8 1/8 0/2 0/10

326 Crop & Pasture Science B. A. Coutts et al.

aphids placed on them at 17 or 31 DAS, 8 of 10 plants werecolonised by 2 DAI with 1–2 aphids/plant, and BWYV wasdetected in 2 of 10 plants in each experiment. When aphidswere placed at 17or 31DASonplants grown fromundressed seedand left without foliar insecticide application, 7 or 8 of 10 plantswere colonized by 2 DAI with 1–2 aphids/plant, and BWYVwasdetected in 5 (Expt 4a) or 2 (Expt 4b) of 10 plants.

Discussion

This study used field exposure followed by aphid inoculation inpot experiments under controlled environment conditions toshow that resistance to BWYV transmission by M. persicaevectors (infection resistance) is present in Australian B. napuscvv. Stubby, Tranby and Trigold. This resistance was not dueto M. persicae feeding preference as aphid colonisation wassimilar in each of them to what it was in BWYV susceptible cv.Pinnacle. Cultivar Banjo apparently also had the same kind ofBWYV infection resistance on field exposure but was not studiedusing aphid inoculations in pot experiments. Cultivars Jade andTanami were ranked as moderately resistant on field exposurewhile the 12 other B. napus genotypes evaluated were ranked assusceptible. The B. napus genotypes that became infected variedwidely in sensitivity to infection with BWYV. This study alsoidentified the cause of the poor performance of imidacloprid seeddressing when applied commercially at the recommended rateto control colonisation by M. persicae and BWYV spread inB. napus stands. This was because the amount of insecticideadhering to seeds was often inadequate to prevent M. persicaecolonisation and BWYV transmission to plants that grew fromtreated seed.

When Thomas et al. (1990) exposed 14 forage Brassicacultivars belonging to 4 different species to infection withBWYV in the field, they ranged from resistant to highlysusceptible (2–99% incidence), with no correlation betweenthe numbers of M. persicae found on the different cultivarsand their susceptibility to infection. B. napus cv. Ramon wasmoderately resistant (20% infection). In our field exposureexperiment, 2 B. napus cultivars (Tranby and Trigold) wereranked as highly resistant to BWYV (0% infection) and2 others (Banjo and Stubby) as resistant (2–5% infection), butmost others were moderately resistant or susceptible. Nosymptoms were observed in the few plants of cvv. Banjo andStubby that became infected, and there was no relationshipbetween susceptibility and sensitivity rankings amonggenotypes ranked moderately resistant or susceptible.

However, as aphid numbers were not assessed in the field itwas unclear whether lack of BWYV infection in cvv. Tranby andTrigold and minimal infection in cvv. Banjo and Stubby was dueto host plant resistance to aphid feeding rather than resistance tothe virus. This question was resolved by pot experiments undercontrolled environment conditions (Expt 1a and 1b) in whichM. persicae showed no cultivar preference when colonising cvv.Stubby, Tranby and Trigold or BWYV-susceptible cv. Pinnacle.These pot experiments were designed so that plants of eachcultivar had equal exposure to aphids flying from a BWYV-infected ‘source’ plant placed centrally, none of them weretouching and the pots were placed on a wire grill to avoid thepossibility of aphids walking from one pot to the next. However,despite all plants of the 4 cultivars being colonisedby aphids, veryfew plants of cvv. Stubby, Tranby or Trigold became BWYV-infected despite all plants of cv. Pinnacle becoming infected.Thus, differences in aphid feeding preference and infestation didnot contribute to the differences in BWYV infection foundbetween cultivars.

The extent of infection resistance in a particular genotype isestablished by inoculating plants with different numbers ofviruliferous aphids as resistance to virus transmission byvector aphids can be overcome if the inoculum dosage(i.e. number of viruliferous aphids) is sufficiently large. Thisform of resistance is well known with the luteovirus Potato leafroll virus (PLRV; family Luteoviridae, genus Polerovirus) inpotato and is quantitative, i.e. polygenically inherited, rather thanbeing determined by single resistance genes (e.g. Wilson andJones 1993a, 1993b; Smith and Barker 1999). In our potexperiments, when different numbers of viruliferous aphidswere placed on B. napus plants, some of cv. Pinnacle alwaysbecame infected with BWYV regardless of the numbers ofviruliferous aphids used to inoculate them (1–10/plant), a totalof 45 out of 68 plants becoming infected. In contrast, out of 68plants of each only 1 of cv. Trigold, 3 of cv. Tranby and 6 of cv.Stubby became infected with BWYV. Thus, these 3 cultivarshave infection resistance to BWYV. In subsequent studiesinvolving pot tests under controlled environment conditions,when 5 plants each of B. napus cvv. 44C73, Hyola50, Opal,Outback, Rocket and Warrior had 5 viruliferous M. persicaeplaced upon each of them before being killed with an aphicide24 h later, 4–5 plants of each cultivar became infected withBWYV (E. Gadja, W. Burton and B. Coutts, unpubl.). Thus,no BWYV infection resistance was found in them. Moreover,cvv. Rocket and Warrior had both been ranked as susceptible in

Table 5. Effects of imidacloprid seed dressing and foliar application either alone or combined on survival ofMyzus persicae and Beet western yellowsvirus infection in Brassica napus cv. Grace (Expt 3a–d) and cv. Beacon (Expt 4a and 4b)

The seed dressing used was Gaucho® at 240 g a.i./100 kg seed and the foliar insecticide was Confidor 200SC® (2 g a.i./L water) applied at 12 h after aphidswere initially placed on plants. Aphid inoculations were at 18, 15, 32 and 29 days after sowing (DAS) for Expt 3a–d; and 17 and 31 DAS for Expt 4a and 4b.Number of plants with surviving aphids counted 2–5 days after inoculation. 10 plants were used for each treatment in each experiment. Petiole samplesfrom all plants were tested individually for BWYV by TBIA at 14 to 33 days after inoculation. Number of plants found BWYV-infected in parentheses

Seed

dressingFoliar

insecticideExpt 3a Expt 3b Expt 3c Expt 3d Expt 4a Expt 4b

Yes Yes 0 (2) 0 (3) 0 (1) 0 (5) – –

Yes No 4 (8) 6 (7) 8 (10) 9 (8) 8 (2) 8 (2)No Yes 0 (2) 0 (5) 0 (3) 0 (4) – –

No No 10 (8) 10 (4) 10 (10) 10 (7) 8 (5) 7 (2)

Control of Beet western yellows virus in Brassica napus Crop & Pasture Science 327

our field experiment with 62% and 57% infection, respectively.Both these findings and our results from pot studies indicatethat using 5 viruliferous aphids/plant to inoculate B. napusgenotypes under controlled environment conditions couldprovide a simple and rapid method to screen B. napusgenotypes for potential infection resistance to BWYV. Notonly would few aphids/plant be required for this asM. persicae is highly efficient at transmitting BWYV(Schliephake et al. 2000), but also only a brief feeding period(<1 h of inoculation access) is required to transmit it (Kyriakouet al. 1983). Such a method would allow initial screening of alarge number of B. napus genotypes for BWYV infectionresistance before field evaluation of a reduced number ofgenotypes. Moreover, the 4 cultivars we found with BWYVinfection resistance could be used as parental lines to helpintroduce this resistance into new B. napus cultivars. Also,these resistant genotypes could be used as reference resistantlines for comparison purposes in future new genotype screeningfor BWYV infection resistance. No clues as to the source ofthe infection resistance were provided by examining theparentage of these 4 cultivars (Table 1). This is consistent withpolygenic inheritance of the infection resistance, as occurs withPLRV (e.g. Wilson and Jones 1993a, 1993b; Smith and Barker1999), but not with inheritance determined by singleresistance genes.

In our pot experiments, imidacloprid seed dressing applied atthe recommended rate (240 g a.i./100 kg seed) using commercialprocedures did not protect 68% of B. napus plants frombecoming infested by aphids or 83% of them developingBWYV-infection (Table 5; Expt 3a–d). In contrast, in thesame experiments foliar application of imidacloprid 12 h afterplacing viruliferous aphids on plants successfully preventedaphid colonisation regardless of presence or absence of theseed dressing and only 28% (seed dressing and foliarapplication) or 35% (foliar application only) of plantsbecame infected with BWYV. In these same experiments, inthe absence of seed dressing or foliar application withimidacloprid, 100% of plants became infested by aphids and72% of them became infected with BWYV. In two further potexperiments in which a small-scale chemical seed applicatorwas used to apply the seed dressing and a different cultivar wasused (Expt 4a and 4b), 15–16/20 plants became infested withaphids regardless of presence or absence of the seed dressingand BWYV was detected in 4/20 (+ seed dressing) or 7/20(– seed dressing) plants. Plant age when aphids were first placedon them did not alter the proportion of plants colonised or thenumbers that became infected by BWYV, so no delay inmovement of the insecticide from treated seed to growingplants was apparent in these experiments. The fewer plantsinfected overall with BWYVwith cv. Beacon (Expt 4a, 4b) thancv. Grace (Expt 3a–d) might possibly reflect a difference inrelative susceptibility between the two. In the field exposureexperiment, cv. Beacon was ranked as susceptible to BWYV,but cv. Grace was not included as it is no longer usedcommercially.

The main conclusion from all 6 pot experiments withinsecticides was that the failure of imidacloprid seed dressingapplied at the recommended rate to control aphid colonisationand BWYV spread effectively was because the amount of

insecticide adhering to most seeds was inadequate. Thisconclusion explains the results of earlier field experiments inwhich this seed treatment applied commercially at therecommended rate was relatively ineffective at preventingcolonisation by M. persicae and transmission of BWYV inB. napus plots (Coutts and Jones 2005; Jones et al. 2007).Possible explanations for the limited success of this seeddressing in controlling M. persicae and virus infection whenused at the recommended rate include (i) the commercialapplication process may not be thorough enough to ensure thatall seeds become coated uniformly with sufficient insecticidefor it to be effective, despite a pink coloured coating being visibleon all of them; or (ii) the recommended rate (240 g a.i./100 kgseed) is insufficient whereas a higher rate (525 g a.i./100 kg)applied by mixing in a glass jar was effective (Jones et al.2007); (iii) there may be inhibitors (e.g. oils or acids) in theseedcoat that prevent the adequate uptakeof the insecticidewhichdiminishes its effectiveness (Halmer 2000) ; or (iv) combinationsof (i)–(iii).

Management of BWYV in B. napus crops requires an IDMapproach that incorporates several diverse control measuresthat operate in different ways (Jones 2004, 2006; Maling et al.2010). These measures include removal of BWYV reservoirhosts (e.g. R. raphanistrum weeds) by herbicide application;manipulating sowing dates to avoid exposure of youngB. napus plants to peak aphid vector flights; sowing at highseed rates to use high plant density to help diminish the rate ofvirus spread; and insecticide application to control aphid vectorpopulations (Jones 2004, 2006). However, further research isneeded to establish effective commercial application proceduresand recommended rates before use of imidacloprid seed dressingcan be recommended. Our research also found that AustralianB. napus cultivars with useful infection resistance to BWYV areavailable. Host resistance can therefore be added to the IDMcontrol measure package. It should not be used alone as, whendeploying cultivars with infection resistance, there is a need toalso limit the numbers of colonising aphid vectors as with highinoculum pressure this type of virus resistance is likely to berelatively ineffective.

Acknowledgments

We thank M. A. Kehoe, B. E. Gadja, and M. Banovich, and staff at MedinaResearchStation for technical assistance. TheAustralianGrainsResearch andDevelopment Corporation provided financial support.

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Manuscript received 17 September 2009, accepted 4 March 2010

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