610 Plant Disease / Vol. 82 No. 6

Pathogenesis of Tomato Spotted Wilt Virus in Peanut PlantsDually Infected with Peanut Mottle Virus

K. Hoffmann, S. M. Geske, and J. W. Moyer, Department of Plant Pathology, North Carolina State University,Box 7616, Raleigh 27695

Tospoviruses cause major diseases ofpeanut worldwide (38). Tomato spottedwilt tospovirus (TSWV) has become in-creasingly important in the production ofpeanut (Arachis hypogaea) in the south-eastern United States. Severe damage topeanut caused yield reductions of up to95% in Texas in 1986, 1990, 1991, and1992 (5,31,32). Although economic lossesin peanut production are less pronounced inother peanut-growing areas, reports of fieldsurveys and anecdotal information indicatean increasing incidence of TSWV in Geor-gia in 19891990 (13), Alabama (25,27),Florida, Mississippi, and North Carolina.

Programs to prevent further spread ofTSWV by insecticidal control of its thripsvector did not sufficiently reduce TSWVincidence (32,44,45). Several managementtools have been recommended for minimiz-ing crop damage due to this virus (6,10,23). Other investigations have focused onthe use of less susceptible varieties (4,15,26) and breeding for disease resistance (14,16,37).

Another widespread virus on peanut,peanut mottle potyvirus (PMV), was firstdescribed in 1965 (29). Its transmission byseeds is assumed to be the reason for itsworldwide distribution in peanut (7) and in

other important crops, such as soybean(Glycine max), pea (Pisum sativum), andbean (Phaseolus vulgaris) (11). The pre-dominant strain of PMV in the UnitedStates is a mild strain (19,34), which causesa faint mottling of the youngest leaves andlittle or no reduction in vegetative plantgrowth (29). Field surveys indicate thepresence of several other strains causingmore severe symptoms, including the ne-crosis (N), necrosis /chlorosis (NC), chlo-rotic stunt (CS), and chlorosis (C) strains(3,30,33,43). The symptoms caused byPMV strains N, C, and CS in peanut aresometimes similar to those due to TSWV(30,42). In addition to the similarity ofsymptoms caused by these viruses, mixedinfections by PMV and TSWV have beenreported in naturally infected peanuts inGeorgia (24,42).

Infection by two unrelated viruses iscommon in higher plants and sometimesleads to a synergistic disease reaction. Inparticular, combinations of a potyvirus andan unrelated virus are known to cause syn-ergistic disease reactions, the best-studiedexample being the interaction between po-tato virus Y and potato virus X (8,17,22,39). Mixed infections often result in a pro-nounced increase in disease severity overthat induced by each virus separately (2,12), sometimes leading to a severe reduc-tion in plant growth and yield losses (35,40). Frequently the titer of the potyvirus indually infected plants is similar to that insingly infected plants, while the titer of theunrelated virus is significantly higher thanin singly infected plants (2,8,12,21,36,41).

The role of PMV in the disease complexvaries, depending on the co-infecting virus.PMV has a synergistic reaction with cu-cumber mosaic virus in cowpea (18) but noeffect in combination with bean pod mottlevirus in soybean (2).

In this study we determined the potentialrole of PMV in the pathogenesis of TSWVin peanut plants dually infected with thesetwo viruses. Experiments were conductedwith six peanut cultivars to reveal the in-fluence of host genetic background on theinteraction.

MATERIALS AND METHODSViruses. A TSWV isolate collected in

1991 from peanut in North Carolina and aPMV isolate that originated from infectedpeanut collected in 1994 in Georgia wereused in this study. The authenticity of theisolates was confirmed by enzyme-linkedimmunosorbent assay (ELISA) and West-ern blot using specific antiserum and byhost range studies. TSWV was maintainedin peanut (A. hypogaea NC7), Nicotianatabacum Burley 21, and Emilia sonchi-folia. PMV was maintained in peanut only.Both viruses were transferred to N. ben-thamiana for one passage to obtain highlyconcentrated inoculum for mechanical in-oculation of peanut.

Virus inocula were prepared from 7- to8-week-old N. benthamiana plants infectedwith TSWV or PMV. Systemically infectedleaves of these plants were triturated 1:5(wt /vol) in 0.01 M Tris, pH 7.8, containing0.01 M Na2SO3 and 0.1% cysteine hydro-chloride. All inoculations were made byrubbing Carborundum-dusted leaves withinoculum-soaked cotton wool applicatorsticks. For inoculations with both virusesin combination, inocula of the two viruseswere mixed 1:1 (vol /vol) before inocula-tion. In all experiments a control of mock-inoculated plants was included.

Peanut cultivars. The peanut geno-types GK7, NC7, VC1, Florunner, South-ern Runner, and Spanco were propagatedfrom seeds. The seeds were pregermi-nated for 2 days in plastic trays contain-ing Metro-Mix (Scotts-Sierra Horticul-tural Products, Marysville, Ohio) andthen were transferred to 4-in. clay pots.The plants were grown, two plants perpot, in a 1:2 mixture of Metro-Mix andsoil, containing fertilizer (888). Theplants were kept under greenhouse con-ditions with natural light only, at tem-peratures between 20 and 30C.

ABSTRACTHoffmann, K., Geske, S. M., and Moyer, J. W. 1998. Pathogenesis of tomato spotted wilt virusin peanut plants dually infected with peanut mottle virus. Plant Dis. 82:610-614.

Peanut (Arachis hypogaea) plants dually infected with tomato spotted wilt tospovirus (TSWV)and peanut mottle potyvirus (PMV) exhibited a wide variety of symptoms, ranging from PMV-like symptoms of transient mild leaf mottle to TSWV-like symptoms of severe leaf distortionand stunting of the plant. Dual infection did not cause greater symptom severity than infectionwith either virus alone. In the early stages of disease development, PMV symptoms were simi-lar to the first leaf symptoms of TSWV infection, suggesting that identification of TSWV infield-grown peanuts should not depend on visual observation. The virus titer, determined byenzyme-linked immunosorbent assay, indicated a delay in TSWV disease progress in double-infected plants, compared to plants infected with TSWV alone. In the later phase of diseaseprogress, the virus titer in dually infected plants was not significantly different from that ofsingly infected plants. Infection with TSWV and PMV alone and with both viruses in combina-tion was consistent among commercially grown peanut cultivars. In plants inoculated withTSWV or PMV alone or with both viruses in combination, the length of the latent period andfinal disease incidence, as measured by the number of plants showing symptoms, did not dependon the cultivar.

Corresponding author: J. W. MoyerE-mail: james_moyer@ncsu.edu

Accepted for publication 2 February 1998.

Publication no. D-1998-0319-01R 1998 The American Phytopathological Society

Plant Disease / June 1998 611

Experimental design. The experimentswere conducted in a split-plot design with1012 plants of each cultivar. All experi-ments were repeated three times at differ-ent seasons of the year. For the split-plotdesign, each experiment was divided intothree blocks, and in each block the culti-vars were randomly assigned to differentrows. In the analysis of the data, samplesof each cultivar and treatment combinationin each block were combined. The blocksare considered replicates in each experi-ment. With one exception, there was nointeraction between the cultivar and thevirus or viruses used for inoculation. There-fore, comparisons of treatments were aver-aged over the cultivars.

Synergism experiments. Symptoms in-duced by TSWV and PMV in dually in-fected plants were compared with thosecaused by these viruses individually. Whenplants had developed two or three leaves,1014 days after planting, all leaflets of asingle quadrifoliate leaf of each seedlingwere inoculated. The same inoculumsource was used for dual and single inocu-lations. The titer of TSWV and PMV inthe two youngest fully or nearly fully de-veloped quadrifoliate leaves of singly anddually inoculated plants was monitored byELISA at various intervals from 8 to 47days after inoculation.

Evaluation of the latent period. Thereactions of the peanut cultivars to TSWVand PMV were monitored after inoculationwith either virus singly or with both vi-ruses simultaneously. The number of plantsshowing TSWV symptoms was determinedat 2-day intervals after observation of thefirst symptoms. Plants showing symptomson at least one leaf were designated symp-tomatic. Disease progress curves were con-structed for each cultivar, using the numberof plants with virus symptoms. Symptomexpression and severity in singly and du-ally infected plants were compared.

ELISA. To monitor the antigen titer ofTSWV and PMV in singly and dually in-

fected peanut plants, an indirect ELISAwas performed. Leaf samples were pre-pared in 1:100 coating buffer (0.05 M car-bonate buffer, pH 9.6), applied directly tothe microtiter plate, and incubated over-night at 4C. The plates were then incu-bated with blocking buffer (0.01 M phos-phate buffer, containing 0.1 M NaCl and1% bovine serum albumin, pH 7.4) over-night at 4C. TSWV- or PMV-specific anti-bodies, diluted in wash buffer (0.05 MTris-HCl buffer, containing 0.1 M NaCland 0.1% bovine serum albumin, pH 7.3),were then applied for 2 h at 37C. TheTSWV antiserum was obtained in our lab-oratory. The PMV antiserum was providedby John Sherwood, University of Georgia,Athens. For the detection of TSWV, puri-fied immunoglobulin G was used at a con-centration of 0.2 mg/ml. PMV antiserumwas applied at a dilution of 2 105. Sub-sequently, the plates were incubated for 2 hat 37C with alkaline phosphataselabeledgoat anti-rabbit antibodies at 1:3,000 inwash buffer. Absorbance readings were de-termined after incubation of the substrate(1 mg of p-nitrophenyl phosphate in 10%diethanolamine, pH 9.8, per ml) at 37Cfor 1 h (PMV) or 2 h (TSWV). Betweeneach step, the plates were washed at leastthree times with wash buffer.

RESULTSEvaluation of the TSWV and PMV la-

tent period. The first symptoms inducedby TSWV were observed in all cultivars 79 days after mechanical inoculation (Fig. 1,TSWV), followed by a linear progressionof disease incidence over time. There wasno significant difference in the latent pe-riod in different peanut genotypes.

Disease progress after mechanical in-oculation with PMV was uniform in allpeanut cultivars, with the first symptomsbeing expressed 5 days after inoculation(Fig. 1, PMV). There was no evidence ofdifferent reactions to PMV in any of thegerm plasm.

Dually infected plants developed leafsymptoms 59 days after inoculation (Fig.1, TSWV/PMV). All peanut cultivars re-acted uniformly following inoculation withmixed inoculum.

Identification of symptomatic plants byvisual diagnosis was confirmed by ELISA.

In most cultivars, the final level of dis-ease incidence was reached 3 weeks afterinoculation, with 70100% of the inocu-lated plants exhibiting visible symptoms ofTSWV or PMV infection and detectablevirus concentrations.

Symptom expression in singly anddually infected plants. Greenhouse-grownpeanut plants inoculated with TSWV aloneat the two- to three-leaf stage developedtypical TSWV symptoms on all system-ically infected leaves, but not always on allleaflets of the quadrifoliate leaves. Thesymptoms began with chlorotic spots,which developed into concentric rings,sometimes accompanied by chlorosis andbronzing of the leaves (Fig. 2B). Symp-toms in later stages of disease developmentincluded stunting and distortion of leavesin the terminal bud and reduced plantgrowth. The expression and severity ofTSWV symptoms varied, but there was nocorrelation between symptom types andpeanut cultivars.

Plants inoculated with PMV alone ex-hibited leaf mottle and a slight reduction inleaf size. Leaves were generally narrowerthan healthy leaves and had pointed tips.The first symptoms were usually expressedon the first leaves to emerge followinginoculation. Particularly in the early stagesof disease development, plants singly in-fected with PMV exhibited symptoms verysimilar to initial symptoms of TSWV in-fection (Fig. 2A). With further diseasedevelopment the similarity in symptomswas less pronounced. The leaf mottlefaded as the plants aged and often wascompletely masked approximately 1month after inoculation. After recoveryfrom PMV infection, the plants grew in a

Fig. 1. Percentage of plants of peanut cultivars GK7, NC7, VC1, Florunner (FR), Southern Runner (SR), and Spanco exhibiting visual symptoms overtime following mechanical inoculation with tomato spotted wilt virus (TSWV), peanut mottle virus (PMV), or a mixture of both viruses (TSWV/PMV).

612 Plant Disease / Vol. 82 No. 6

manner similar to that of the uninoculatedcontrol.

Plants inoculated with both TSWV andPMV exhibited a wide range of symptoms,including typical TSWV and PMV symp-toms (Fig. 2C and D). New symptomtypes, differing from those of either virus,were not observed. Most of the duallyinfected plants exhibited TSWV-like symp-toms (Fig. 2C). These plants were severelystunted, and all systemically infected leaveswere chlorotic, distorted, and reduced insize. A few doubly infected plants ex-pressed a PMV-like leaf mottle, which laterfaded as the plants recovered (Fig. 2D).

TSWV antigen titer in singly and du-ally infected plants. The TSWV titer inthe youngest fully or almost fully expandedquadrifoliate leaf of singly and doublyinfected peanut plants varied over time(Table 1). In plants singly infected withTSWV, the virus titer reached a detectablelevel 2 weeks after inoculation. The high-

est concentration of antigen was detectedapproximately 3 weeks after inoculation.In dually infected plants TSWV could bedetected after 710 days. For both treat-ments, the peak of TSWV antigen was fol-lowed by a decrease in virus concentration.Up to 3 weeks after inoculation, there weresignificant differences between the virustiter in plants singly infected with TSWVand that in dually infected plants. Duringthis period, the TSWV titer also variedamong the peanut genotypes. In later stagesof disease development, neither the inocu-lated virus or viruses nor the cultivar in-duced significant differences in virus titer.

Besides the nonuniform distribution ofthe virus in a single plant at a specific timeand over the course of disease develop-ment, there were considerable differencesin the TSWV and PMV titers in plants ofthe same cultivar subjected to the sametreatment. The wide variation in titer in in-dividual plants is the reason for the high

standard deviation of the experimentaltreatments (Tables 1 and 2).

In similar experiments conducted withmechanically inoculated N. benthamiana,the same delay in building up a detectableTSWV titer occurred in dually infectedplants, compared with singly infectedplants. TSWV was detected by ELISA 5days after inoculation with TSWV aloneand 10 days after inoculation in duallyinfected plants. In later stages of infectionof N. benthamiana, singly and dually in-fected plants developed the same TSWVtiter (data not shown).

PMV antigen titer in singly and du-ally infected plants. At the beginning ofinfection, the PMV titer was significantlyhigher in singly than in dually infectedplants (Table 2). In later stages of diseasedevelopment, no significant differenceswere observed between the PMV antigentiter in quadrifoliate leaves of singly in-fected plants and that in correspondingleaves of dually infected plants. The PMVtiter varied strongly over time, peakingtwice in both singly and dually infectedplants, 13 and 33 days after inoculation.Since no cultivar treatment interactionsoccurred, comparisons of treatments wereaveraged over the cultivars.

DISCUSSIONIn greenhouse studies, no significant dif-

ferences in disease progress were observedin the peanut cultivars GK7, NC7, VC1,Florunner, Southern Runner, and Spancoafter mechanical inoculation with TSWV,PMV, or both viruses. The period of timerequired for symptom expression and thepercentage infected plants, as measured byvisual assessment of symptoms andELISA, were similar in all cultivars. Withrespect to TSWV, these results differ fromprevious observations. In field-grown pea-nuts subjected to the natural spread of thevirus transmitted by its vector, the finalincidence of spotted wilt was lower anddisease progress was slower in SouthernRunner than in Florunner (15). From ourresults it is concluded that the resistanceobserved in the field is not due to specificresistance against TSWV. Therefore, thisresistance may be effective only at lowinoculum dosage, as has been shown forpeanut bud necrosis tospovirus (20), or itmay be vector dependent.

No synergistic disease reaction was ob-served in plants dually infected with TSWVand PMV. Dually infected plants may ex-hibit symptoms of either virus, with a widerange of symptom severity. Peanut plantsinoculated with TSWV and PMV mainlyexpressed symptoms similar to those in-duced by TSWV alone. In general, symp-tom severity did not increase by co-inocu-lation with TSWV and PMV. Up to 3weeks after inoculation, a delay in diseaseprogress in the presence of PMV was de-tected. In dually infected plants the TSWVtiter reached a detectable level later than in

Fig. 2. Systemically infected leaves of the peanut cultivar GK7, 14 days after inoculation with peanutmottle virus (A) and tomato spotted wilt virus (B), showing similar symptoms, and 14 days after in-oculation with a mixture of the two viruses (C and D), indicating a wide range of symptom types andseverity.

Plant Disease / June 1998 613

plants infected with TSWV only. Duringdisease development, PMV did not inducean increase in the TSWV titer, nor did itincrease symptom severity in dually in-fected plants.

In many cases of infection by a poty-virus and an unrelated virus, the effect onthe host plant is different from that ob-served in this experiment. For example,dual infections with soybean mosaic poty-virus and different comoviruses caused asynergistic disease reaction in soybeanplants, including increases in symptom se-verity and in the virus titer of the como-virus (2,12,40). Similar effects occur incowpea stunt disease, caused by a synergis-tic reaction of cucumber mosaic cucumo-virus (CMV) and blackeye cowpea mosaicpotyvirus (1,35), as well as in combina-tions of CMV with zucchini yellow mosaicpotyvirus (36) and with turnip mosaic poty-virus (41).

For the interaction of potato virus X andpotato virus Y (17,22,39), the molecularmechanisms causing plant viral synergismhave been investigated in detail (8), indi-cating that 5 proximal potyviral sequencesmediate the synergistic disease reaction intobacco (9).

As shown previously by Kresta et al.(28) for naturally infected peanuts, TSWVwas not distributed uniformly throughout theplant, with the highest virus concentration inyoung, developing leaf tissue. The expres-sion of TSWV symptoms was positivelycorrelated with the detection of the virus byELISA. Symptomless leaves of infectedplants and even symptomless leaflets ofleaves with typical TSWV symptoms didnot give positive ELISA readings (28). Wefound no correlation between symptom se-verity and ELISA readings, in contrast to thefindings of Kresta et al. (28).

After infection with TSWV and PMV,peanut plants exhibited a wide range ofsymptoms, including typical symptoms ofeither virus. In addition, particularly in theearly stages of disease development, plantsinfected with PMV exhibited symptomsvery similar to the initial leaf symptoms ofTSWV infection. These results confirmearlier observations of naturally infectedpeanuts in the field (42). Identification ofTSWV in field-grown peanuts shouldtherefore not rely solely on visual detectionof virus symptoms. Laboratory tests arerequired for reliable virus identification asa basis for adequate control measures.

ACKNOWLEDGMENTSThis project was supported in part by a grant

through a cooperative agreement with the Univer-sity of Georgia supported by Goldkist.

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Table 2. Peanut mottle virus (PMV) antigen titer in peanut plants infected with PMV or with PMVand tomato spotted wilt virus (TSWV), as determined by enzyme-linked imunosorbent assay (ELISA)

Days after ELISA valuesb

Standard Treatmentinoculationa PMV PMV + TSWVc error difference 8 0.777 0.385 0.058 P 0.05 13 1.847 1.498 0.044 P 0.05 19 1.158 1.373 0.084 d26 0.288 0.331 0.037 33 0.672 0.882 0.048 P 0.00140 0.611 0.551 0.049 47 0.255 0.303 0.032 a The youngest quadrifoliate leaf was assayed at the times indicated. Leaf extracts were prepared at

1:100 (wt /vol) in carbonate coating buffer (pH 9.6).b Optical density (A405 nm), mean value of 72 plants. No cultivar treatment interaction occurred.

Therefore, comparisons of treatments were made with data averaged over the cultivars. Opticaldensity values of the uninoculated control ranged between 0.01 and 0.03.

c Inocula of the viruses were mixed 1:1 prior to inoculation.d Not significant.

Table 1. Tomato spotted wilt virus (TSWV) antigen titer in peanut plants infected with TSWV orwith TSWV and peanut mottle virus (PMV), as determined by enzyme-linked immunosorbent assay(ELISA)

Days after ELISA valuesb

Standard Treatmentinoculationa TSWV TSWV + PMVc error difference 8 0.014 0.017 0.001 P 0.05 13 0.083 0.017 0.008 P 0.05 19 0.268 0.064 0.014 P 0.05 26 0.284 0.252 0.017 d33 0.222 0.183 0.013 P 0.00140 0.098 0.114 0.012 47 0.115 0.099 0.011 a The youngest quadrifoliate leaf was assayed at the times indicated. Leaf extracts were prepared at

1:100 (wt /vol) in carbonate coating buffer (pH 9.6).b Optical density (A405 nm), mean value of 72 plants. No cultivar treatment interaction occurred.

Therefore, comparisons of treatments were made with data averaged over the cultivars. Opticaldensity values of the uninoculated control ranged between 0.01 and 0.02.

c Inocula of the viruses were mixed 1:1 prior to inoculation.d Not significant.

614 Plant Disease / Vol. 82 No. 6

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