Plant Pathology Laboratory, Faculty of Agriculture, Aristotle University of Thessaloniki, Thessaloniki, Greece

Transmission of Zucchini yellow mosaic virus by Colonizing and Non-colonizing

Aphids in Greece and New Aphid Species Vectors of the Virus

N. I.N. I. KatisKatis11,, J. A.J. A. TsitsipisTsitsipis

22,, D. P.D. P. LykouressisLykouressis33,, A.A. PapapanayotouPapapanayotou

11,, J. T.J. T. MargaritopoulosMargaritopoulos22,, G. M.G. M. KokinisKokinis

11,,

D. Ch.D. Ch. PerdikisPerdikis33 andand I. N.I. N. ManoussopoulosManoussopoulos

22

Authors� addresses: 1Plant Pathology Laboratory, Faculty of Agriculture, Aristotle University of Thessaloniki, 541 24Thessaloniki, Greece; 2Laboratory of Entomology and Agricultural Zoology, University of Thessaly, 384 46 Nea Ionia,Magnesia, Greece; 3Laboratory of Agricultural Zoology and Entomology, Agricultural University of Athens, Iera Odos, 118

55 Athens, Greece (correspondence to N. I. Katis. E-mail: katis@agro.auth.gr)

Received August 5, 2005; accepted January 11, 2006

Keywords: Zucchini yellow mosaic virus, non-persistent transmission, aphid vectors, cucurbits, Greece

AbstractNineteen aphid species were tested for their ability totransmit Zucchini yellow mosaic virus (ZYMV) fromand to zucchini under laboratory conditions. Sixteenspecies were found to be new vectors of ZYMV (i.e.Aphis craccae, Aphis fabae, Aphis nerii, Aulacorthumsolani, Brachycaudus cardui, Brevicoryne brassicae, Hy-alopterus pruni complex, Hyperomyzus lactucae, Mac-rosiphoniella sanborni, Macrosiphum rosae,Metopolophium dirhodum, Myzus cerasi, Rhopalosiphummaidis, R. padi, Semiaphis dauci and Sipha maydis).Their transmission efficiency by a single aphid was low(0.1–4.2%). Myzus persicae was used as a control andwas the most efficient vector (41.1%, one aphid perplant). Hayhurstia atriplicis, Myzus ascalonicus and Si-tobion avenae did not transmit the virus. In four out ofsix new vectors assayed in arena tests for propensityestimation, propensity was higher than efficiency. Datafrom an experimental zucchini field in northern Greecerevealed a high correlation between ZYMV spread andalatae of the vector species. The most abundant aphidvectors during 2 years experimentation were M. persi-cae, Aphis gossypii and Aphis spiraecola. The possiblerole of the 16 new and the previously known aphid vec-tors in the epidemiology of ZYMV was investigatedusing data of transmission efficiency combined with thecaptures of their alatae in the Greek net of a Rotham-sted type suction trap.

IntroductionZucchini yellow mosaic virus (ZYMV; family Potyviri-dae, genus Potyvirus) affects cucurbit crops throughoutthe world (Lisa et al., 1981; Lecoq et al., 1983; Lese-mann et al., 1983; Provvidenti et al., 1984; Namethet al., 1985; Desbiez and Lecoq, 1997). Yield losses incantaloupe were particularly high when infectionoccurred before fruit set (Blua and Perring, 1989).

Similar results were observed in zucchini Cucurbitapepo L. (Cucurbitaceae) when mechanical inoculationwas made during 2–3-weeks period after transplanta-tion in experiments under cover (J. A. Tsitsipis et al.,unpublished data). In Greece, the virus was first recor-ded in southern regions of the country in cucumber,Cucumis sativus L. (Cucurbitaceae) and zucchini crops(Kyriakopoulou and Varveri, 1991). ZYMV has sincebeen regularly isolated from field-grown cucumber,zucchini, melon, Cucumis melo L. and watermelon[Citrulus lanatus (Thunb.)] all over the country and ithas always been associated with considerable crop los-ses (A. Avgelis, personal communication; N. I. Katis,unpublished data). The role of ZYMV has becomemore important in cucurbit production as periodicepidemics have appeared throughout the country (Va-itsopoulos and Katis, 1993; Papavassiliou, 2000).

ZYMV infects cucurbit plants and is transmittednon-persistently by many colonizing and non-colon-izing aphid species (Perring et al., 1992; Desbiez andLecoq, 1997). In this mode of transmission (forreviews see Katis, 1989; Pirone and Perry, 2002)aphids transmit the viruses by short test probes, lastinga few seconds, to evaluate the suitability of the hostplant during the host selection process.

To date 10 aphid species of the family Aphididaehave been reported as vectors of ZYMV: Aphis gos-sypii Glover (Lisa et al., 1981); Aphis craccivora Koch(Adlerz, 1987); Aphis spiraecola Patch (Lisa andLecoq, 1984); Aphis middletonii Thomas (Adlerz,1987); Acyrthosiphon kondoi Shinji (Perring et al.,1992); Acyrthosiphon pisum (Harris) (Adlerz, 1987);Lipaphis erysimi (Kaltebach) (Adlerz, 1987); Macrosip-hum euphorbiae (Thomas) (Lisa and Lecoq, 1984);Myzus persicae (Sulzer) (Adlerz, 1987) and one Uroleu-con species (Perring et al., 1992). Blackman and Eastop(2000) have reported more than 10 aphid species

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J. Phytopathology 154, 293–302 (2006)� 2006 The AuthorsJournal compilation � 2006 Blackwell Verlag, Berlin

colonizing cucurbits including five vectors (A. gossypii,A. craccivora, A. spiraecola, M. persicae and M. euphor-biae). In Greece, surveys in field cucurbit crops revealedthat the major aphid pest is A. gossypii (J. A. Tsitsipiset al., unpublished data). Cucurbits in Greece, how-ever, are usually cultivated near or immediately adja-cent to other crops such as cotton, Gossypium hirsutumL. (Malvaceae), tobacco, Nicotiana tabacum L. (Solan-aceae), vegetables and thus non-discriminatory alight-ing of non-colonizing aphid species or of occasionallyaphid pests cannot be excluded. Although most aphid-borne viruses are assumed to be spread by colonizingaphid species and additional prejudice exists to focuson them (Kennedy et al., 1962), non-colonizing aphidspecies also contribute to increased virus spread ofnon-persistently transmitted viruses especially whenthey land in large numbers on the crops (Swenson,1968; Raccah et al., 1985; Fereres et al., 1993; Perezet al., 1995). Virus transmission efficiency also varieswith various species (e.g. Katis and Gibson, 1984; Cas-tle et al., 1992; Webb and Kok-Yokomi, 1993; Yuanand Ullman, 1996; Nebreda et al., 2004) and compari-sons, therefore, of the relative transmission efficienciesof different aphid species enable the identification ofthe important contributors to virus spread.

Our aim, therefore, was to test whether variousaphid species, not previously reported as vectors, cantransmit ZYMV in classical transmission experimentsand arena tests and to investigate the possible role ofaphid vector abundance in relation to virus spread andepidemiology. Additionally, we wished to determine ifthere is a possible correlation between the aphid popu-lations of colonizing and non-colonizing species andthe ZYMV spread in the field.

Materials and MethodsProduction of apterous aphids

Aphid species were reared on suitable hosts. Plantswith aphid colonies were kept in growth chambersunder controlled conditions (20–25�C, 60 ± 5% relat-ive humidity and photoperiod of 16 h light per day).In the tests, wingless adults starved for 1–2 h, wereused. To synchronize aphid development, so thatadults of approximately the same age were availablefor the experiments, the following procedure wasadopted. Sufficient numbers of young adult winglessaphids were placed on respective host plants and leftfor 2 days for larviposition. They were then transferredto new plants and the procedure was repeated four tofive times. Newly moulted adults were used for theexperiments.

Direct transmission tests

The following 19 aphid species (Hemiptera: Aphidi-dae): Aphis craccae L., Aphis fabae Scopoli., Aphisnerii Boyer de Fonscolombe., Aulacorthum solani(Kaltenbach), Brachycaudus cardui (L.), Brevicorynebrassicae (L.), Hayhurstia atriplicis (L.), Hyalopteruspruni complex, Hyperomyzus lactucae (L.), Macrosi-phoniella sanborni (Gillette), Macrosiphum rosae (L.),

Metopolophium dirhodum (Walker), Myzus ascalonicusDoncaster, Myzus cerasi (F.), Rhopalosiphum maidis(Fitch), R. padi (L.), Semiaphis dauci (F.), Sitobion ave-nae (F.) and Sipha maydis Passerini were examined inthe direct transmission tests and M. persicae was usedas a control. In all experiments, aphids were allowed3–5 min acquisition access to diseased plants and thentransferred and caged on single healthy test plants ingroups of 30, whereas M. persicae, because of itsexpected high transmission efficiency, was caged singly.Next day, plants were sprayed with imidacloprid to killaphids and then kept in a glasshouse for 3–4 weeks forsymptom development. Transmissions were made fromand to zucchini plants (cv. Jedida F1) at the cotyledonstage. For each transmission experiment 10–12 plantswere used and the procedure was repeated five to 10times.

Arena tests

Transmission of ZYMV by six aphid species(A. fabae, A. nerii, A. solani, B. brassicae, R. maidisand R. padi) was examined in arena tests. One zuc-chini plant, infected with the virus 2 weeks earlier,was positioned in the middle of five healthy plantsarranged on a circle, and covered by insect-proof netcages. Thirty wingless adult aphids from each aphidspecies were placed on the zucchini diseased plantand left for 24 h. Next day, plants were sprayed withimidacloprid and placed in a glasshouse for symptomdevelopment. Similar treatments were carried out withM. persicae as controls placing only one aphid on theinfected plant. For each aphid species a total of 65–100 plants were tested.

Monitoring of aphid populations

Data on the occurrence and population fluctuation ofwinged aphids were obtained from captures of aphidsfrom Rothamsted type suction traps located in fiveeconomically important agricultural areas: uplandLasithi, Crete; Koroivos, western Peloponnese; Farmof the Agricultural University of Athens, Kopaida,Voiotia; Farm of the University of Thessaly, Veles-tino, Magnesia and Farm of the Aristotle Universityof Thessaloniki, Thessaloniki during 1995–1998(Fig. 1). In addition, aphid populations were monit-ored in a zucchini field (0.1 ha) in Vassilika, Thessalo-niki (northern Greece) by two Moericke yellow watertraps from June to August 1995 and from August toOctober 1997. In 1995, the crop was planted in 10June, whereas in 1997 early August. Winged aphids,caught in both suction and yellow traps, were storedin plastic vials containing one volume of lactic acid(75%) and two volumes of ethyl alcohol (95%) untiltheir identification. Aphids were identified accordingto the keys of Jacky and Bouchery (1980), Taylor(1984), Brown (1989) and Remaudiere and SecoFernandez (1990). Furthermore, in the same zucchinifield, ZYMV spread was monitored by weekly sam-plings of 100 randomly selected, regardless of virussymptoms, zucchini leaves.

294 Katis et al.

Virus identification

In all cases, virus identification was based on enzyme-linked immunosorbent assay (ELISA) according toClark and Adams (1977) using polyclonal antibodiesprovided by H. Lecoq (INRA, Montfavet, France).The concentration of c-globulin immunoglobulin (IgG)used was at 1 lg/ml and the conjugate was diluted1000 times.

ResultsTransmission of ZYMV

In the direct transmission tests, 16 of the 19 aphid spe-cies tested transmitted a local isolate (Vassilika, nor-thern Greece) of ZYMV, at a different efficiency(Table 1) and they are reported as vectors for the firsttime. Their one aphid-transmission probability, as cal-culated by the formula described by Gibbs and Gower(1960), ranged from 0.07 to 4.15, while the mean trans-mission efficiency for M. persicae, using one aphid perplant, was 41.07 (Table 1). Transmission (%) was sig-nificantly different among vector species (X2

18 ¼ 489.2,P < 0.001). The most effective ones were A. nerii,A. craccae, B. cardui, R. padi and M. sanborni withcorresponding transmission efficiencies 72%, 60%,55%, 54% and 48%. A second group of vectors, A. fa-bae, A. solani, M. rosae and S. dauci, had transmissionefficiencies ranging from 14% to 25% whereas theother vectors had very low transmission efficiencies (2–5%; Table 2). Hayhurstia atriplicis, M. ascalonicus andS. avenae did not transmit the virus. The one aphid-transmission probability ranks the aphid species vectorin the same order.

In arena tests (Table 1) transmission (%) differedsignificantly among the aphid species examined(X2

5 ¼ 39.1, P < 0.001). The transmission efficiency ofthe aphid species A. nerii, R. padi, A. fabae and A. sol-ani ranged from 47% to 60%, while B. brassicae andR. maidis transmitted the virus less efficiently (34%and 10%, respectively). Comparing the results of directtransmission and arena tests, A. nerii transmitted thevirus at a significantly higher percentage in the directtest (X2

1 ¼ 8.4, P < 0.004), while M. persicae

Fig. 1 Location of suction traps in Greece

Table 1Transmission of Zucchini yellow mosaic virus to zucchini plants by different aphid species in direct transmission and arena tests and oneaphid-transmission probability

Aphid speciesDirect

transmissionsaOne aphid

probability (%)bM. persicae control indirect transmissiona Arena testa

M. persicae controlin arena testa

Aphis nerii 72 (50) a 4.15 34 (100) 47 (100) ab 48 (100)Aphis craccae 60 (100) a 3.00 40 (100) – –Brachycaudus cardui 55 (100) a 2.60 42 (100) – –Rhopalosiphum padi 54 (112) a 2.60 52 (112) 60 (100) a 55 (100)Macrosiphoniella sanborni 48 (100) a 2.15 42 (100) – –Aphis fabae 25 (112) b 0.95 52 (112) 55 (65) ab 62 (65)Aulacorthum solani 17 (100) b 0.61 35 (100) 52 (100) ab 46 (100)Macrosiphum rosae 15 (100) b 0.54 40 (100) – –Semiaphis dauci 14 (100) b 0.50 37 (100) – –Hyperomyzus lactucae 5 (100) c 0.17 36 (100) – –Myzus cerasi 5 (100) c 0.17 37 (100) – –Hyalopterus pruni 4 (100) c 0.14 35 (100) – –Metopolophium dirhodum 4 (100) c 0.14 38 (100) – –Brevicoryne brassicae 4 (112) ce 0.12 52 (112) 34 (100) b 45 (100)Rhopalosiphum maidis 3 (112) ce 0.09 52 (112) 10 (100) c 50 (100)Sipha maydis 2 (100) ce 0.07 42 (100) – –Hayhurstia atriplicis 0 (100) de 0.00 40 (100) – –Myzus ascalonicus 0 (100) de 0.00 34 (100) – –Sitobion avenae 0 (100) de 0.00 36 (100) – –Myzus persicae 41.07 41 (1948) 50 (565)

aPercentage infected plants, number in brackets denote number of tested plants.bThe one aphid probability calculated according to the formula of Gibbs and Gower (1960).–, not performed.Numbers in each column followed by a different letter differ significantly (P < 0.05) by chi-square test.

295Transmission of Zucchini yellow mosaic virus by Colonizing and Non-colonizing Aphids in Greece

(X21 ¼ 15.3, P < 0.001), A. fabae (X2

1 ¼ 16.5, P <0.001), A. solani (X2

1 ¼ 27.1, P < 0.001), B. brassicae(X2

1 ¼ 30.7, P < 0.001) and R. maidis (X21 ¼ 4.9,

P < 0.03) showed significantly higher percentagetransmission in the arena test. By contrast, R. padishowed a similar transmission efficiency (X2

1 ¼ 0.7,P < 0.42) in both tests.

Monitoring of winged aphids

The total number of winged individuals and aphid spe-cies caught in the four suction traps during the survey-ing period is shown in Table 2. Differences in bothtotal number of individuals and aphid species amongregions and years were observed. The highest totalnumber of winged individuals as well as of aphid spe-cies was recorded in Thessaloniki. At least in threeregions (i.e. Thessaloniki, Velestino and Kopaida), thehighest number of winged aphids was captured in1997. In all regions the average over the years monthlycaptures of winged individuals of aphid vectors followmore or less these of winged individuals of all aphidspecies (Fig. 2). A significant number of the totalwinged aphids and aphid vectors was recorded duringthe growing season of Cucurbitaceae (late-March tolate-November in southern locations and from May toOctober in central and northern ones). However, somedifferences were observed in the pattern of captures,especially in the peaks, among regions.

From an epidemiological point of view apart fromthe fluctuation of the population of aphid vectors therelative importance of each aphid vector species andthe possible risk of infection during the cultivated per-iod are also concerned. In this direction, we attempt toestimate a vector pressure index (VPI; DiFonzo, 1995),i.e. the portion of aphids of each vector that couldtransmit the virus after landing on a plant. This indexwas calculated according to the equation: number ofspecies vector · one aphid-transmission probability.We used our data of the one aphid-transmission prob-ability for the 15 of 16 new vectors found in the pre-sent study and for the known vectors we used datafrom other published papers. For M. persicae we useddata obtained in this study and also data publishedelsewhere. When more than one value was found inbibliography for a given species, then the mean oneaphid-transmission probability was used.

Tables 3 and 4 show the total number of wingedindividuals of aphid vectors captured in each year aswell as the estimated number of winged aphids that

could transmit the virus after landing on a plant.Twenty-three vector species were found in Greece dur-ing the surveying period, 23 in Thessaloniki, 22 inVelestino, Kopaida and Koroivos and 20 in Lasithi. Itappears that six aphid species (A. gossypii, M. persicae,A. pisum, A. craccivora, R. padi and A. spiraecola)have shown the highest VPI values and thereforemight be of major importance in the epidemiology ofZYMV (Tables 3–5). These high VPI values could beattributed mainly to the high one aphid-transmissionprobability that these species exhibit and to a lesserextend to the high number of winged aphid captured,as in the case of R. padi, which has the lower oneaphid-transmission probability among these six vec-tors. The one aphid-transmission probability of theseaphid vectors are 0.35 for M. persicae (calculated fromdata of this study, Lisa et al., 1981; Purcifull et al.,1984; Adlerz, 1987; Castle et al., 1992), 0.32 forA. craccivora (calculated from data of Yuan and Ull-man, 1996), 0.27 for A. spiraecola (calculated fromdata of Purcifull et al., 1984; Adlerz, 1987), 0.18 forA. gossypii (calculated from data of Castle et al., 1992;Yuan and Ullman, 1996), 0.11 for A. pisum (calculatedfrom data of Castle et al., 1992) and 0.03 for R. padi(calculated from our data).By contrast, some aphid vectors such as H. pruni

and M. dirhodum, which reached high populations(1167 and 807 mean number of winged aphids cap-tured in all regions and years) showed extremely lowmean VPI values (2 and 1) due to their low transmis-sion efficiency (Table 1). Differences were alsoobserved in total VPI values between surveying yearswithin each region. In all regions, except Kopaida, theincrease in the total VPI value in certain years was dueto outbreaks of A. gossypii and M. persicae. In Kopa-ida, the high VPI values that were recorded in 1997and 1998 were due to an enormous increase in the cap-tures of the alatae of A. pisum and also of R. padi inthe latter year.Figure 2 shows the temporal fluctuation of the aver-

age over all years VPI values of colonizing and non-colonizing aphid species in each region. A generaltrend was that colonizing aphid species showed higherVPI values than non-colonizing aphids. This is due tothe fact that colonizing aphids (e.g. A. gossypii,M. persicae) showed high populations and/or the relat-ively high transmission efficiency rates. In three regions(Thessaloniki, Kopaida, Koroivos) high VPI valueswere observed during a wider period of time, while in

Table 2Total number of alatae and aphidspecies (in brackets) captured inRothamsted type suction trapsin Greece during 1995–1998

Site

Sampling year

1995 1996 1997 1998 1999

Thessaloniki –a 28143 (145) 41851 (162) 16974 (122) 7745 (109)Velestino 2594 (145) 12780 (129) 14053 (125) 2509 (65) 4333 (86)Kopaida 7694 (129) 15576 (142) 24154 (120) 21341 (106) –a

Koroivos –a 7528 (130) 6866 (121) 7756 (119) 4279 (108)Lasithi –a –a –a 2179 (86) 2674 (72)

aData not available.

296 Katis et al.

Lasithi and Velestino only for a 2-month period. Themonthly fluctuation of VPI values (pooled data fromcolonizing and non-colonizing aphid species) over all

years does not correlated well with the fluctuation ofcaptures of alatae of aphid vectors in all regions (Pear-son’s correlations; Thessaloniki: N ¼ 48, r ¼ 0.582,P < 0.001; Velestino: N ¼ 60, r ¼ 0.972, P < 0.001;Kopaida: N ¼ 48, r ¼ 0.844, P < 0.001; Koroivos:N ¼ 48, r ¼ 0.627, P < 0.001; Lasithi: N ¼ 24, r ¼0.973, P < 0.001). This could be explained by the factthat certain aphid species with low transmission effi-ciency (e.g. H. pruni) appeared in large numbers whileothers with high transmission efficiency (e.g. M. persi-cae) were captured in low numbers.

Spread of ZYMV in the field

Forty-three and 46 aphid species and 1350 and 1308winged aphids per Moericke trap were captured in theexperimental zucchini field in 1995 and 1997 respect-ively. Of the aphid species captured, 13 species vectorsof ZYMV were found in 1995 and 17 in 1997. Theprevailing aphid species were M. persicae (69% and34% of the total captures in 1995 and 1997, respect-ively), A. gossypii (13% and 9%, respectively) andA. spiraecola (2% and 17%, respectively), all efficientvectors of ZYMV. In both years, high Pearson’s corre-lation coefficients between the cumulative number ofwinged individuals of total aphid species and percent-age ZYMV infection were found (N ¼ 9, r ¼ 0.957,P < 0.001 in 1995 and N ¼ 4, r ¼ 0.993, P < 0.007in 1997). Percentage of ZYMV infection was also cor-related well with the cumulative number of wingedaphids of vector species and VPI (N ¼ 9, r ¼ 0.957,P ¼ 0.001 and N ¼ 9, r ¼ 0.959, P < 0.001 in 1995and N ¼ 4, r ¼ 0.987, P < 0.013 and N ¼ 4, r ¼0.988, P < 0.012 in 1997, respectively; Fig. 3).

DiscussionNon-persistently transmitted aphid-borne virusesspread rapidly often causing epidemics with great los-ses in different crop plants including cucurbits. Tounderstand the dynamics of such epidemics and todevelop management strategies, knowledge on the vec-tor composition, their relative abundance and theirpotential capability for virus transmission is veryimportant (Difonzo et al., 1997). ZYMV has been dis-tributed all over the world rapidly during the last twodecades. In Greece, ZYMV incidence in zucchini cropshas increased substantially over time presumably dueto the abundance of virus sources and/or the numberof aphid species involved. These have been supportedexperimentally in the current study in two field experi-ments with zucchini in Vassilika, Thessaloniki duringthe cultivating period of 1995 and 1997. In these

Fig. 2 Fluctuation of mean vector pressure index values (over allsampling years), i.e. number of alatae of species vectors · one aphidprobability transmission (VPI-NC and VPI-C for non-colonizing andcolonizing aphid species, respectively), and mean number of wingedaphids of total and vector species caught in Rothamsted type suctiontraps in (a) Thessaloniki, northern Greece and Velestino, centraleastern Greece, (b) Kopaida, central Greece and Koroivos, southernGreece and (c) Lasithi, Crete. Solid lines denote the growing periodof zucchini in each region

297Transmission of Zucchini yellow mosaic virus by Colonizing and Non-colonizing Aphids in Greece

Table 3Total number of alatae of vector species captured (N) in suction Rothamsted type traps and vector pressure index (VPI ¼ number of alataeof each species captured · one aphid probability transmission) in Thessaloniki, northern Greece and Velestino, central eastern Greece

Aphid species

Thessaloniki Velestino

1996 1997 1998 1999 1995 1996 1997 1998 1999

N VPI N VPI N VPI N VPI N VPI N VPI N VPI N VPI N VPI

Acyrthosiphon pisum 477 50 488 52 189 20 155 16 283 30 56 6 556 59 13 1 61 7Aphis craccae 2 0 387 12 13 0 2 0 5 0 0 0 12 0 2 0 9 0Aphis craccivora 70 23 248 80 119 38 150 48 250 80 221 71 247 79 126 40 107 34Aphis fabae 59 1 127 1 67 1 45 0 85 1 148 1 218 2 14 0 10 0Aphis gossypii 4369 767 699 123 530 93 592 104 15 436 2709 3366 591 4126 724 1138 200 2081 365Aphis nerii 80 3 31 1 22 1 27 1 17 1 9 0 10 0 1 0 12 1Aphis spiraecola 30 8 95 26 47 13 39 11 6 2 66 18 111 30 24 7 49 14Aulacorthum solani 113 1 252 2 126 1 72 0 34 0 119 1 213 1 0 0 5 0Brachycaudus cardui 14 0 16 0 20 1 3 0 59 2 90 2 511 13 4 0 4 0Brevicoryne brassicae 38 0 130 0 95 0 129 0 96 0 30 0 28 0 6 0 8 0Hyalopterus pruni 2426 3 3133 4 6766 10 1362 2 237 0 1419 2 852 1 39 0 479 1Hyperomyzus lactucae 38 0 239 0 74 0 11 0 38 0 59 0 165 0 6 0 8 0Lipaphis erysimi 0 –a 153 0 30 – 32 – 61 – 11 – 51 – 2 – 7 –Macrosiphoniella sanborni 3 0 0 0 0 0 0 0 2 0 0 0 2 0 0 0 0 0Macrosiphum euphorbiae 75 –a 772 0 149 – 33 – 254 – 495 – 59 – 8 – 55 –Macrosiphum rosae 0 0 0 0 25 0 27 0 1 0 4 0 15 0 0 0 4 0Metopolophium dirhodum 0 0 9947 14 1952 3 738 1 42 0 161 0 670 1 14 0 17 0Myzus cerasi 34 0 25 0 18 0 11 0 15 0 12 0 10 0 2 0 0 0Myzus persicae 274 96 280 98 107 37 55 19 109 38 227 79 272 95 20 7 69 25Rhopalosiphum maidis 486 0 338 0 167 0 116 0 378 0 332 0 137 0 36 0 137 0Rhopalosiphum padi 954 25 1932 50 697 18 220 6 394 10 1365 36 783 20 35 1 98 3Semiaphis dauci 0 0 17 0 0 0 1 0 0 0 0 0 0 0 0.0 0 0 0Sipha maydis 5 0 18 0 2 0 6 0 0 0 0 0 2 0 0.0 0 1 0Total 9547 977 19 327 463 11 215 235 3826 209 17 802 2873 8190 808 9050 1028 1490 256 3221 447

aNot adequate data found in literature.

Table 4Total number of alatae of vector species captured (N) in suction Rothamsted type traps and vector pressure index (VPI ¼ number of alataeof each species captured · one aphid probability transmission) in Kopaida, central southern Greece, Koroivos, southern Greece and Lasithi,Crete, Greece

Aphid species

Kopaida Koroivos Lasithi

1995 1996 1997 1998 1996 1997 1998 1999 1998 1999

N VPI N VPI N VPI N VPI N VPI N VPI N VPI N VPI N VPI N VPI

Acyrthosiphon pisum 65 7 346 37 4613 488 2033 215 144 15 237 25 109 12 199 21 112 12 38 4Aphis craccae 0 0 19 1 0 0.0 8 0 27 1 33 1 94 3 33 1 102 3 2 0Aphis craccivora 116 37 56 18 206 66 329 106 197 63 124 40 298 96 95 31 8 3 33 11Aphis fabae 5 0 7 0 13 0 8 0 56 1 92 1 60 1 18 0 29 0 55 1Aphis gossypii 607 107 382 67 892 157 290 51 950 167 550 97 629 110 191 34 34 6 77 14Aphis nerii 6 0 0 0 5 0 2 0 3 0 3 0 9 0 2 0 1 0 0 0Aphis spiraecola 0 0 0 0 1 0 18 5 68 18 96 26 122 33 18 5 1 0 2 1Aulacorthum solani 24 0 97 1 77 1 28 0 155 1 86 1 51 0 35 0 28 0 12 0Brachycaudus cardui 2 0 5 0 24 1 5 0 27 1 22 1 7 0 8 0 5 0 3 0Brevicoryne brassicae 3 0 7 0 7 0 18 0 7 0 7 0 3 0 3 0 101 0 79 0Hyalopterus pruni 311 0 981 1 681 1 485 1 670 1 2226 3 667 1 831 1 61 0 127 0Hyperomyzus lactucae 16 0 27 0 29 0 18 0 85 0 81 0 35 0 9 0 8 0 7 0Lipaphis erysimi 3 –a 18 – 19 – 87 – 185 – 52 – 238 – 15 – 3 – 2 –Macrosiphoniella sanborni 0 0 2 0 1 0 0 0 1 0 1 0 0 0 0 0 0 0 0 0Macrosiphum euphorbiae 6 –a 160 – 25 – 18 – 306 – 157 – 46 – 23 – 12 – 53 –Macrosiphum rosae 1 0 11 0 12 0 6 0 0 0 18 0 5 0 8 0 3 0 5 0Metopolophium dirhodum 11 0 310 0 820 1 1282 2 119 0 91 0 65 0 8 0 20 0 13 0Myzus cerasi 6 0 6 0 13 0 11 0 44 0 11 0 87 0 58 0 44 0 16 0Myzus persicae 122 43 447 156 494 173 115 40 307 107 543 190 810 283 62 22 29 10 1392 487Rhopalosiphum maidis 896 1 259 0 256 0 130 0 354 0 98 0 1736 2 30 0 20 0 21 0Rhopalosiphum padi 201 5 476 12 407 11 4570 119 828 22 358 9 300 8 81 2 131 3 6 0Semiaphis dauci 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0Sipha maydis 3 0 5 0 11 0 2 0 4 0 4 0 0 0 0 0 0 0 0 0Total 2404 200 3621 294 8606 898 9463 539 4537 397 4890 393 5371 548 1727 117 752 38 1943 516

aNot adequate data found in literature.

298 Katis et al.

experiments, the ZYMV incidence in both years washighly correlated well with the captures of wingedaphid vectors indicating their significance in the virusspread. Relationships between winged aphid popula-tions of various species and virus spread have alsobeen established for other Potyviruses, e.g. lettuceLactuca sativa L. (Compositae) and Brassica crops-Lettuce mosaic virus (LMV; family Potyviridae, genusPotyvirus; Nebreda et al., 2004); potato Solanum

tuberosun L. (Solanaceae)-Potato virus Y (PVY; familyPotyviridae, genus Potyvirus; Katis et al., 1998); pep-per Capsicum annum L. (Solanaceae)-PVY, Cucumbermosaic virus (CMV; family Bromoviridae, genusCucumovirus; Raccah et al., 1985). However, other fac-tors (e.g. virus sources; Eastop and Raccah, 1988) mayalso be involved in non-persistent virus epidemics thanthe increase in vector numbers. Further research isneeded towards (e.g. surveying for weeds hosts ofZYMV) that direction for a better understanding ofZYMV epidemics in Greece.

Ten aphid species had previously been reported asZYMV vectors and 16 new aphid vectors have beenfound in the present study of which two are cucurbitcolonizers (A. fabae and A. solani) and 14 non-coloniz-ers (Blackman and Eastop, 2000). From the previouslyknown vector species M. persicae (Lisa et al., 1981;Purcifull et al., 1984; Adlerz, 1987; Castle et al., 1992),A. craccivora (Yuan and Ullman, 1996), A. spiraecola(Purcifull et al., 1984; Adlerz, 1987), A. gossypii (Yuanand Ullman, 1996) and A. pisum (Castle et al., 1992)are the most efficient, whereas, the rest seem to trans-mit the virus inefficiently. The data of direct transmis-sion tests presented in this study showed that the 16new aphid vectors transmit the virus (one aphid-trans-mission probability 0.07–4.15) much less efficientlythan the aforementioned species and the parthenoge-netic lineage of M. persicae (one aphid-transmissionprobability 0.41) used here as control. In the arenatests at least for some virus/vector combinations pro-pensity was lower than efficiency. Aphis nerii transmit-ted the virus in much higher percentages in direct thanin arena tests whereas no differences were observedbetween the tests in the case of R. padi. A lower pro-pensity than efficiency has also been reported in experi-ments with A. gossypii and A. craccivora (Yuan andUllman, 1996). By contrast, in five aphid species(M. persicae, A. fabae, A. solani, B. brassicae andR. maidis) transmission in arena tests was significantlyhigher than in direct tests. Our data support the hypo-thesis of Irwin and Ruesink (1986) that different viewsof vector importance could be provided when vectorefficiency and propensity is examined. These differencescould be attributed to the nature of the arena methodwhich allows a candidate vector more behaviouralfreedom in probing duration and frequency as well as

Table 5The most important aphid species in the Zucchini yellow mosaic virus (ZYMV) epidemiology according to mean vector pressure index (MVPI)which were captured in suction Rothamsted type traps in the five regions surveyed

Vector species Mean Thessaloniki Velestino Kopaida Koroivos Lasithi

Aphis gossypii 279 (1591) 272 (1548) 918 (5229) 95 (543) 108 (580) 10 (56)Myzus persicae 123 (351) 63 (179) 49 (139) 103 (295) 151 (431) 248 (711)Acyrthosiphon pisum 54 (507) 35 (327) 21 (194) 189 (1764) 18 (172) 8 (75)Aphis craccivora 46 (143) 47 (147) 61 (190) 57 (177) 57 (179) 7 (21)Rhopalosiphum padi 17 (672) 25 (951) 14 (535) 37 (1414) 10 (392) 2 (69)Aphis spiraecola 10 (37) 14 (53) 14 (51) 1 (5) 21 (76) 1 (2)Total 536 (6086) 471 (10 979) 1083 (7951) 483 (6024) 364 (4131) 277 (1348)

See Table 1 for details regarding the years that the traps operated. Numbers in brackets denote mean number of alatae captured.

0

10

20

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40

50

60

70

80

90

100

22Jun

25Jun

28Jun

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27Jul

31Jul

3Aug

6Aug

10Aug

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enta

ge (

%)

of in

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lant

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INF ATSAV VPI1995

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14 Aug 21 Aug27 Aug 3 Sep 10 Sep17 Sep24 Sep 1 Oct 8 Oct 15 Oct

Perc

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INF ATSAV VPI

1997

Fig. 3 Incidence of Zucchini yellow mosaic virus (ZYMV; INF) inzucchini plots, cumulative vector pressure index (VPI ¼ number ofalatae of aphid vectors · one aphid probability transmission) andcumulative number of alatae of total aphid (ATS) and vector species(AV) captured in Moericke yellow traps in Vassilika, Thessaloniki in1995 and 1997

299Transmission of Zucchini yellow mosaic virus by Colonizing and Non-colonizing Aphids in Greece

the opportunity to visit more plants. A further explan-ation is that A. solani, B. brassicae and R. maidis arenon-colonizers of zucchini (Blackman and Eastop,2000) and presumably zucchini it is not a preferablehost for A. fabae and M. persicae. Field surveysrevealed that A. gossypii is the most important aphidpest of zucchini in Greece (J. A. Tsitsipis et al., unpub-lished data). Thus, more aphids of these species pre-sumably move from the infected zucchini plant andasses a higher number of the surrounded non-infectedplants. However, this hypothesis does not hold forA. nerii and R. padi examined in the present study aswell as for A. gossypii and A. craccivora examined byYuan and Ullman (1996) in a similar system and forthree aphid vectors of Beet yellows virus (BYV; familyClosteroviridae, genus Closterovirus) non-colonizingsugar beet (Summers et al., 1990). It seems, therefore,that other factors are also involved in the differentcombinations of vectors/viruses/host plants, and thisphenomenon needs further investigation given thatsuch information will be of great importance for thedevelopment of forecasting schemes.

According to some authors (Summers et al., 1990;Yuan and Ullman, 1996) the arena method, whichmeasures vectors propensity, is a better way to evalu-ate vector importance than the direct transmissiontests which measure efficiency. In five of seven vectorsspecies examined in this study the propensity washigher than their transmission efficiency implying ananalogous behaviour under natural conditions. Never-theless, taking into account that the results of arenatests did not alter the rank of vectors provided bydirect transmission tests, our results regarding trans-mission efficiency of the new vectors found in the pre-sent study along with those for the known vectorspublished in other papers could give insights in theepidemiology of ZYMV in Greece.

It has been proposed that inefficient vectors mayplay an important role to the epidemiology of non-per-sistent viruses, provided that they appear in high pop-ulations. For example, it has been suggested that R.padi, an inefficient vector of PVY, might play animportant role in virus epidemiology in the UK, whereit appears in high populations compared with the mostefficient vector M. persicae which appeared in low pop-ulations (Katis and Gibson, 1985). In addition, Uroleu-con pseudambrosiae (Olive) (Hemiptera: Aphididae), aninefficient vector of Watermelon mosaic virus (WMV;family Potyviridae, genus Potyvirus) is presumablyinvolved in the spread of this virus in Florida (USA)due to its high populations (Webb and Kok-Yokomi,1993). Nevertheless, this does not hold in every crop/non-persistent virus system. For instance, Ioannou(1989) found that PVY incidence in potato crops waspoorly correlated with total number of flying aphidsbut well with the number of winged individual of M.persicae which is an efficient colonizing species. In ourstudy the suction trap data showed that H. pruni and M.dirchodum, both non-colonizing species, appeared inhigh populations in almost all regions and years. How-

ever, their very low transmission efficiency and theirrespective mean VPI (2 and 1, respectively) questionstheir importance in ZYMV epidemiology.However, the calculated VPI has some limitations.

We use transmission efficiency data obtained in differ-ent laboratories where different experimental protocolswere applied. Also, parameters regarding the tendencyof the aphids to land and to probe on the crop shouldbe examined and incorporated in the calculations.Thus, field observations with green tile traps (Irwin,1980) to estimate the landing rate of aphid species onzucchini plants should be conducted in the future.Towards that direction data on ZYMV spread inmany zucchini plots are needed in order to investigatefor correlations with the abundance of specific aphidspecies (Madden et al., 2000) in suction traps. Thiswould probably further clarify the role of inefficientaphid vectors in the spread of ZYMV. Nevertheless,our approach gives some valuable information,although with the some limitations, on ZYMV epi-demiology in Greece.In the zucchini experimental fields in northern

Greece, ZYMV incidence and spread was highly corre-lated well with three parameters examined, i.e. totalnumber of winged aphids, aphid vectors and VPI. Itseems, however, that the three efficient vectors(M. persicae, A. gossypii and A. spiraecola) colonizingzucchini (Blackman and Eastop, 2000) play a majorrole in ZYMV spread as they appear in large numbers(84% and 60% of the total alatae captured in 1995and 1997, respectively). By contrast, inefficient vectorssuch as H. pruni and M. dirhodum, which were cap-tured in high numbers in suction traps, appeared invery low numbers in Moericke traps (1–7 wingedaphids per trap). The aforementioned three vectorswere among the six ones that the combined data ofsuction traps and one aphid probability transmissionefficiency suggested that they are presumably import-ant in spread of ZYMV (Table 5). It is worth mention-ing, however, that data from more zucchini fields inother regions with diverse climatic conditions and cropcultivations systems are needed in order to furtherclarify the importance of different virus vectors.Spread of ZYMV was correlated well with totalwinged aphid captures in zucchini fields. Thus, from apractical point of view this parameter could giveimportant information for virus occurrence in cucurbitcrops. Nevertheless, additional work is needed for totalwinged aphid captures to be used as a risk index, asthe case of high populations of inefficient vectors, non-colonizing cucurbits and low ZYMV incidence couldnot be excluded. Furthermore, prediction schemescould not be based only on suction traps data, whichprovide information on a large regional scale andcould not account for aphid population on certainfields and localities. Comparing the captures of thesuction trap in Thessaloniki and Moericke traps inVassilika (5 km from Thessaloniki) in 1997, differencesare revealed regarding the prevailing species. In Moer-icke traps M. persicae, A. gossypii and A. spiraecola

300 Katis et al.

(60% of the total alatae captured) were the prevailingspecies whereas in the suction trap the species M. dir-hodum, H. pruni and R. padi (36% of the total alataecaptured). These three prevailing species in Moericketaps presented the 3% of the total captures in the suc-tion trap. Thus, data from Moericke traps are alsoneeded for a better understanding of the ZYMVepidemiology and for more accurate forecastingschemes at least at a small regional scale. Suctiontraps, however, provide information for wide areasand long-range risk. More data are needed for theevaluation of possible correlation between the twotrapping systems.

Cucurbits are cultivated from late-March to late-November in southern Greece and from May to Octo-ber in central and northern Greece. In most locationszucchini is cultivated continuously, in two or three suc-cessive crops, a practical that usually leads to virusepidemics of the middle (summer) to late season crops.This is due to cumulative virus sources in the previousand usually heavily affected cucurbit crops. Our resultsshow that there is a continuous risk of ZYMV infec-tions due to the presence of different aphid vectorsduring the whole growing season. They also suggesthigher risk of infection at certain periods during thegrowing season, i.e. mainly May and July–August andalso October–November in the southern regions. Manyaphid vector species of ZYMV found in the present orin previous studies do not colonize cucurbits. Swenson(1968) suggested that in the case of non-colonizingaphids, non-discriminatory alighting and dominance ofdispersal over host finding coupled with duration andintensity of migration contributes to an increase ofvirus spread. In addition, non-colonizing aphids due totheir mobility through the crops they are rarely targetsof pesticide spraying programmes. Thus, measureswhich prevent aphid landing (e.g. reflective and strawmulches, bait plants, row covers) could be more effect-ive against non-colonizing species reducing virusspread at the initial stages of the crop where is moresusceptible to virus infection (Blua and Perring, 1989;J. A. Tsitsipis et al., unpublished data). Thus, takinginto account the fluctuation of population of aphidvectors and that of VPI, planting of zucchini cropsearly in May could decrease the losses caused byZYMV, due to the lack of synchronization betweenhigh aphid populations and high susceptibility of zuc-chini to the virus. Furthermore, the growers should bein alarm when adjacent crops are at harvest (e.g.wheat in June) or heavily infested by aphids (e.g. cot-ton where large populations of A. gossypii are fre-quently observed), because massive dispersal of alateaphids is expected. From these point of view zucchinicrops should not be planted in the vicinity of othersthat harbour aphid vectors of ZYMV. Also, oil appli-cation at periods of high risk, after field evaluation,could also be an alternative.

To conclude, in the present study 16 new aphid vec-tors of ZYMV have been identified raising the total ofvectors species to 26. The new vectors are less efficient

than some of those previously known but someappeared in large numbers in suction traps and theymight play a role in ZYMV epidemiology. However,field experiments in one location in northern Greeceshowed that the most abundant aphids were M. persi-cae, A. gossypii and A. spiraecola, known efficientvectors that colonize cucurbits. Data form suctiontraps established throughout the country revealed thatduring the whole growing season there is a continuouspresence of aphid vectors, although higher risk ofinfection exists presumably in May, July–August andOctober–November according to the region and year.Infections during the latter period, however, are notcausing serious crop losses as the plants are alreadymature and therefore less vulnerable to virus infection.The fact that the majority of the aphid species testedin the present study (16 of 19) transmitted the virussuggests that the virus may easily be transmitted bymany species and it is quite possible that many otheraphid species could also be vectors of ZYMV. Moreresearch is needed towards that direction. Given thattransmission efficiency values help in the developmentof simulation models and that intraspecific variation isan important factor, research on genetic divergentpopulations is important to be examined. Finally,additional field experiments are needed combining dif-ferent trapping methods such as suction, Moericke andtile traps.

Acknowledgements

The present study was supported by an EPET II 453 research grant

by the Hellenic General Secretariat for Research and Technology.

The authors thank Dr H. Lecoq, Station de Pathologie Vegetale, BP

94, 84143, Montfavet, France for providing antisera specific for

ZYMV, and Dr V. F. Eastop, Natural History Museum (London)

for aid with aphid identification.

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