Spread of zucchini yellow mosaic potyvirus in squashin Hungary*Z. Basky, T. M. Perring and I. To bia sPlant Protection Institute Hungarian Academy of Sciences, Budapest, Hungary

Abstract: The temporal and spatial distribution of zucchini yellow mosaic potyvirus (ZYMV) was studied in a 3000-m2

zucchini squash ®eld. The ®rst infected plants were found 4 weeks after the ®eld was exposed to virus source plants. The

infection increased to nearly 74% by the end of the study. Alate aphids were active from the beginning of the study and43 species were trapped in the ®eld. Flights of vector species Acyrthosiphon pisum andMyzus persicae peaked during thefourth week which resulted in high virus incidence 4 weeks later. There was a signi®cant correlation between the

number of vectors caught in yellow pan traps and the number of infected plants in the ®eld. In laboratory studiesevaluating 11 aphid species, Aphis pomi de Geer was identi®ed as a new vector species of ZYMV. Although this aphidwas not caught in our ®eld studies, it may be an important contributor in other areas where cucurbits are grown in closeproximity to apple or other hosts of this aphid.

1 Introduction

Zucchini yellow mosaic potyvirus (ZYMV) is a widelydistributed virus that causes devastating epidemics in arange of cucurbit crops. The virus ®rst was described inItaly (LISA et al., 1981). Since then it has spread toFrance, Germany, Israel, Lebanon, Morocco andSpain (LISA and LECOQ, 1984), Great Britain, Turkey,Lebanon, Jordan, Egypt, Japan, Taiwan, Australia,Canada, and the United States (SAMMONS et al., 1989).It occurred in 1983 and 1987±88 in the Netherlands butsystemic removal of diseased plants led to the eradi-cation of the disease (SCHRIJNWERKERS et al., 1991).ZYMV causes serious economic losses in cucurbitcrops in the Central and Southern Vallies of California(PERRING et al., 1992; GRAFTON-CARDWELL et al.,1996). The virus severely a�ects cucurbit crops inSaudi Arabia (ALHUDAIB, 1997). In Hungary ZYMV®rst was detected in 1995 (To BIA S et al., 1996).

ZYMV is e�ciently transmitted by several aphidspecies in a non-persistent manner (LISA and LECOQ,1984). Aphis gossypii Glover was reported as ane�cient vector of ZYMV by LISA et al., 1981. Aphiscitricola (syn.: Aphis spireacola Van der Goot), Aphismiddletonii (Thomas), Myzus persicae (Sulzer), Lypa-phis erysimi (Kaltenbach), Aphis craccivora Koch,Acyrthosiphon pisum (Harris) and Uroleucon sp. thatwere caught alive e�ciently transmitted ZYMV inlaboratory transmission tests (ADLERZ, 1987). Acyr-thosiphon kondoi Shinji and Rhopalosiphum padiproved to be vectors of ZYMV in a ®eld transmissiontest (CASTLE et al., 1992).

The objective of the present study was to monitor theprogress of ZYMV incidence in the ®eld given a singleinternal virus source. Virus incidence was documentedin time and space in relation to the aphid ¯ight andvector activity. In addition, aphid species that arecommon to Hungarian agriculture were evaluated aspotential vectors of ZYMV.

2 Materials and methods

2.1 Temporal distribution

Zucchini seeds of the variety `Vitamin' were sown withspacings 1 m between rows and between seeds. One hundredplants were sown in 30 rows creating an experimental ®eld of3000 m2. To study the spread of ZYMV the initial internalvirus source was established by placing 12 mechanicallyinoculated potted zucchini plants in the middle of the plot (atthe 50th plant of the 15th row) when the zucchini emerged inthe ®eld. The source plants were inoculated 2 weeks beforethey were placed in the ®eld, and they were replaced by newinoculated plants every 2 weeks. Twenty-®ve healthy pottedzucchini plants in the cotyledon stage were placed at the 30thplant of the 10th row for a 1-week exposure period to monitorthe virus infection pressure. Each week, from emergence ofzucchini in the ®eld until the end of the experiment, theindicator plants were replaced with new ones. After exposurethese indicator plants were kept in an aphid-free greenhousefor 4 weeks for symptom development. Plants were tested forCMV and ZYMV using an enzyme-linked immunosorbentassay (ELISA) (CLARK and ADAMS, 1977).

2.2 Spatial distribution of ZYMV

The ®eld was monitored once each week for the number andlocation of infected plants, which were labelled. Plants were*This paper is dedicated to Dr Ja nos Szirmai for his 90th birthday.

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J. Appl. Ent. 125, 271±275 (2001)Ó 2001 Blackwell Wissenschafts-Verlag, BerlinISSN 0931-2048

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considered to be infected when the characteristic symptomswere noticed (mottling, leaf malformation, stunting, mosaicand dark green blisters); all such plants were tested for thepresence of ZYMV using ELISA. The number of newlyinfected plants for each week was used to test di�erencesbetween sampling dates using t-test (STATSOFT, 1994).Twenty randomly selected sampling locations, each locationconsisting of 12 plants (three sequential plants in fouradjacent rows) were established as sampling units in the ®eld.These locations were used to test the di�erences of virusincidence between sampling dates and sampling locations byusing an analysis of variance (STATSOFT, 1994). Yatescorrected v2 test was used to show di�erences betweencumulative virus infection at the various sampling locationswithin each sampling date (STATSOFT, 1994).

2.3 Aphid abundance and disease incidence

A Moericke yellow water pan trap was placed at the 60thplant of the 20th row to monitor alate aphid ¯ight. Aphids inthe trap were collected twice a week, and identi®ed using astereo-microscope using the keys of BLACKMAN and EASTOP

(l984), BASKY (1993) and TAYLOR (1984). Virus incidence ofthe ®eld plants was correlated with the number of aphidscaught by the trap 4 weeks prior to virus assessment to allowsymptom development in the ®eld plants.

2.4 Aphid transmission: laboratory study

The ability of 11 aphid species to transmit ZYMV wasdetermined in laboratory studies. We evaluated Aphis pomide Geer, Aphis hederae Kalt., Brachycaudus helichrysi Kalt.,Chaitophorus vitellinae Schrank, Macrosiphum rosae L.,Myzus persicae Shulzer, Rhopalosiphum padi (L.) Metopol-ophium dirhodum (Walker), Sitobion avenae (Fabricius),Schizaphis graminum (Rondani) and Diuraphis noxia (Mor-dvilko). Some of the species were collected in the ®eld andcolleagues were reared in the laboratory. Aphis pomi wascollected from commercial apple, A. hederae from Hederahelix, B. helichrysi from commercial plum trees andC. vitellinae from Salix sp. Myzus persicae was reared onChinese cabbage and the other species on barley.Potted cucumber plants in the cotyledon stage were

mechanically inoculated with ZYMV. These plants servedas the virus source 2 weeks after inoculation. The test plantsalso were potted cucumber seedlings in the cotyledon stage.Two hours prior to transmission tests, aphids were removedfrom their respective cultures, and con®ned in Petri dishes fora starvation period. After starvation apterae were placedwith a ®ne camel hairs brush on the virus source for a 5 minacquisition access period. Following acquisition, 10 aphidswere transferred to a single test plant. In this manner, 10 testplants were infested with each species. Twenty-four hourslater plants were sprayed with PIRIMOR 50 DP (Zeneca,UK)1 aphicide and placed in an aphid-free greenhouse forsymptom development. Four weeks after inoculation theplants were assessed visually and with ELISA for ZYMVinfection.

3 Results and discussion

3.1 Temporal distribution of ZYMV

Symptoms of virus infection ®rst were observed4 weeks after the source plants were placed in the®eld. At this time, only two of the 3000 ®eld plants

(0.06%) showed clear ZYMV symptoms. The percent-age infection steadily increased during the studyresulting in 63.43% ®eld infection by the eighth weekof the study. Virus infection of the indicator plantsoccurred ®rst during the third week of the study. Thevirus infection percentage of the indicator plantsbeginning from the third week varied between 15.4and 24.8% during the experimental period (®g. 1).ELISA testing the indicator plants revealed 54% of theplants were infected with both ZYMV and CMV. Dueto the continuous presence of the internal ZYMVsource, ZYMV predominated. The ELISA showed thepresence of ZYMV in 92.5% of infected indicatorplants, and 61.5% of the plants were infected withCMV. The high proportion of CMV in the indicatorplants was due to the fact that CMV is the mostfrequent virus disease of Cucurbitaceae plants inHungary, and it is considered to be one of the mostimportant pepper viruses (To BIA S et al., 1982; BASKY

and NASSER, 1989; BASKY and RACCAH, 1990).Analysis of variance showed that ZYMV incidence

increased signi®cantly during the time in the samplinglocations (F � 56.86 P � 0.0000). The ®rst ZYMVsymptom was observed 4 weeks after emergence ofzucchini plants. There were no signi®cant di�erences(P � 0.318) in ZYMV incidence between weeks 4 and 5(table). However the disease epidemic resulted insigni®cantly more plants becoming infected betweenweeks 5 and 6 (P � 0.001). New plants became infectedat the same rate between weeks 6 and 7 (P � 0.35). Inweek 8, the ®nal week of the study, a signi®cant(P < 0.0001) increase in new infections occurred. Theinfection reached 74% in the sampling locations. Asimilar tendency was observed by ADLERZ (1987) inwhich infection dramatically increased 3 weeks afterthe ®rst symptoms were recorded in the ®eld.

3.2 Spatial distribution of ZYMV

There were no visible signs of infection in the whole®eld for the ®rst 3 weeks of the study. On week 4,location 9 had a single plant with symptoms. It isinteresting to note that the ®rst symptom-showingplant was located 31.8 m from the source plants, whichwere placed in the centre of the ®eld. Despite thisrelatively large distance from the source plants, we arecon®dent that the ZYMV originated from the central

Fig. 1. Virus infection pressure based on indicator plantinfection percentage

272 Z. Basky, T. M. Perring and I. To bia s

internal virus source, because the virus was not presentelsewhere in the region.

The most important vectors of ZYMV were tran-sient Acyrthosiphon pisum and Myzus persicae in ourstudy. These aphid species do not colonize squash soafter probing on the non-host virus source, theirsettling response was inhibited and ¯ight was resumed.This may have resulted in longer ¯ight (cf. 31.8 m).Yates-corrected v2 analysis indicated that there wereno signi®cant di�erences (P > 0.05) in virus infectionbetween the 20 sampling locations on week 5. Howeveron week 6, statistical di�erences were present suggest-ing an aggregated distribution. Virus incidence of 14locations with zero or one infected plants di�ered(v2 � 5.56, P � 0.018; v2 � 9.19 P � 0.024) from loca-tions with more than one infected plant. There wasfurther statistical separation between locations 2, 3, 6,and 8 infected plants (v2 � 4.29, P � 0.038).

One of the locations (location 9) is interesting, sincethis was the location where the ®rst plant in the ®eldwas infected on week 4: just 2 weeks later eight of the12 sample plants were infected. This suggests that thepoint source infection had spread to surroundingplants. This also indicates that the virus was acquiredfrom the point source 2 weeks before characteristicZYMV symptoms developed. Two weeks earlier mildmosaic symptoms indicated the virus infection, but atthis early stage the ZYMV symptoms were similar tothose of CMV. It took two further weeks to developthe characteristic symptoms of ZYMV infection (leafnarrowing, blistering and malformation). Plants wererecorded as infected by ZYMV when these severesymptoms were visible.

The aggregated distribution in location 9 also couldbe due to vector activity of colonizing Aphis gossypii.Unlike transient vectors this species may ¯y or evenwalk for shorter distances (1±2 m) from one host to thenext and transmit the virus. This is supported by the®ndings of MORA-AGUILERA et al., 19922 ): absence ofaggregation of papaya ringspot virus was due to lackof colonizing aphids.

On week 7, 11 of the 20 sampling locations had atleast one plant with virus symptoms. Fifteen of thelocations had two or fewer plants with virus and ouranalysis indicated that these 15 were statisticallydi�erent from the ®ve locations with more virus(v2 � 5.56, P � 0.018). Virus disease continued tospread at location 9, as 11 of the 12 plants showedvirus symptoms. The disease incidence at this locationwas signi®cantly higher than all the plots, with theexception of one other location. It is interesting to note

that the density of infected plants in the close vicinityof the central virus source was not higher than anyother place of the ®eld. In fact, of the 12 plants at thislocation, the ®rst one became infected on week 5. Onweek 6, the second plant became infected, and six newinfections occurred on week 8. This location had eightinfected plants by the end of the study.

The incidence of disease on week 8 was markedlyhigher than the previous weeks and all locations hadinfected plants. One location had 25% infection; thiswas signi®cantly di�erent from other locations wherevirus infection varied between 41 and 100%.

Integrating the spread of disease across the seasonshows a pattern which is characteristic of non-persist-ently transmitted pathogens. Early in the season, thelack of di�erences between the plots re¯ects the lowincidence of disease present in the ®eld and suggests arandom distribution of virus.

Later in the season di�erences between the locationsindicated aggregated distribution of the virus, and bythe time of the last monitoring the plots had becomeevenly infected; 74% of the plants showed symptoms.Our results are in agreement with that of FERERES

et al. (1992) in that early ZYMV infections wererandom. As the virus spread, diseased plants becamemore aggregated around the early point-source infec-tions, and ®nally the entire ®eld was uniformlyinfected. These data agree with the results of MADDEN

et al. (1987) who suggested that non-persistentlytransmitted tobacco etch and tobacco vein mottlingvirus was distributed randomly at the beginning of theseason, and became aggregated at the end of theepidemic. THRESH (1974) also noted this to be astandard pattern of distribution.

3.3 Aphid abundance and disease incidence

Between 13 June and 16 August, 864 individuals of 38species and ®ve genera were caught in the yellow pantrap. Species of genus Aphis sp. was the most numer-ous genera comprising 21.9% of the total catchfollowed by Acyrthosiphum pisum (Harris) 15.9% andMetopolophium dirhodum (Walker) 13%. Althoughlarge Aphis gossypii colonies were present on 10% ofthe zucchini plants the proportion of Aphis gossypii didnot reach 5% in the yellow pan trap. This is typical, ascolonizing aphids have little tendency to ¯y.

Two peaks of aphid ¯ight were recorded. The ®rstpeak occurred during week 4, and the second duringweek 7. To evaluate the relationship among vectorsand virus incidence we conducted correlation analyses

Table 1. Average propor-tion of newly infested plantsin the sampling locations foreach week of the study

Sampling dateInfested plants

(weeks) Number mean � SEM d.f. t P

0±3 0 0.000 � 0.0004 1 0.004 � 0.002 a 2395 4 0.016 � 0.008 a 239 )1.0 0.3186 18 0.075 � 0.001 b 239 3.58 0.0011

7 22 0.092 � 0.019 b 239 0.928 0.358 131 0.545 � 0.035 c 239 12.93 0.0001

1Means � SEM followed by di�erent letters are signi®cantly di�erent (P < 0.001).

Spread and aphid transmission of ZYMV 273

between total number of aphids trapped and virusincidence, and between numbers of known ZYMVvectors and virus incidence. We followed the methodsof MORA-AGUILERA et al. (1992) who found that therequired time lag between aphid abundance andsymptom expression of papaya ringspot virus was 1month. Although mild mosaic symptoms were visible2 weeks after infestation it took another 2 weeks todevelop characteristic ZYMV symptoms (malforma-tion, leaf narrowing and blistering). Plants wererecorded to be infected when these severe ZYMVsymptoms occurred; therefore the required time lagbetween aphid abundance and ZYMV symptomexpression was 1 month.

Our analyses showed that disease incidence in the®eld was signi®cantly correlated with total aphidnumbers 1 month earlier (r � 0.96, P < 0.05). Theproportion of aphids made up of species known totransmit ZYMV was 20.5% of the total catch. Whenwe used correlation analysis on the known vectorspecies (A. craccivora, A. gossypii, A. pisum, L. erysimi,M. persicae, and R. padi) the relationship was nearly asstrong as with total aphid numbers (r � 0.93,P < 0.05). This suggests that the majority of virusspread was related to the abundance of the knownvector species (®g. 2), and it is doubtful that otherspecies trapped in the study were involved in the virusspread. Regarding individual aphid species, there was asigni®cant correlation between number of A. pisum andM. persicae and ZYMV incidence in the ®eld 1 monthlater (r � 0.65 and 0.59, respectively, P < 0.05) Aphisgossypii did not play a prominent role in ZYMVtransmission in our experimental ®eld early in theseason, because trap numbers were low, becoming highonly by the end of the season. We expect, however,that A. gossypii was responsible for ZYMV transmis-sion which resulted in aggregated virus distribution inthe ®eld, and for transmissions found outside the ®eld.We observed an increase in A. gossypii numbers in theyellow pan, which was likely due to senescence ofthe experimental plants. This emigration resulted in theZYMV infection occurring in another squash ®eldlocated at a distance of 100±120 m from the experi-mental ®eld. PERRING et al. (1992) suggested thecolonizing vector aphids must be induced to leave the

host plant before they contribute to virus spread. Inthe present study A. gossypii stayed on the squash untilthe end of the growth cycle. At this time they canbecame a vector of virus from the ®eld.

Some ZYMV-infected squash plants were foundoutside the experimental ®eld after the study, but therewas no sign of ZYMV infestation on cucurbit plants inthe region during the next season. Although one of thehosts Lamium amplexicaule L. is a widely distributedweed in Hungary, ZYMV did not seem to over winterin the region, presumably because other cucurbitswhich play prominent role in overwintering of the virus(PERRING et al., 1992) are not present in the Hungarian¯ora. However, the present study demonstrates that ifZYMV becomes established in a squash ®eld, the viruscan spread rapidly with the aphid species that occur inthe region.

3.4 Aphid transmission test

Our studies identi®ed a new vector of ZYMV; 30% ofthe test plants were infected when Aphis pomi was usedas the vector. Although it was not found in our ®eldstudy, A. pomi is widespread in Europe, in the MiddleEast and it has been reported from North America. Itcolonizes apple, pears, quince and several other generaof woody Rosaceae such as Chaenomeles, Cotoneaster,Crataegus, Mespilus and Sorbus (BLACKMAN andEASTOP, 1984). Although the species is monoecious, itis found regularly in yellow pans in pepper, potato andcucumber ®elds in Hungary. Aphis pomi could beimportant in areas where cucurbit crops are grown inclose proximity to apple orchards and other hosts ofthe species. In our studies, this aphid was not caught atour ®eld site.

Fifty per cent of the test plants became infected in thelaboratory when M. persicae was tested. With the samemethod, 30% of the test plants showed symptoms ofZYMV when R. padi was the vector. Rhopalosiphumpadi has been found to transmit ZYMV in the ®eld, butnot in the laboratory because alate did not probe oninfected squash source plants (CASTLE et al., 1992).

Schizaphis graminum, and S. avenae did not transmitZYMV in our experiment. These results con®rm thoseof ADLERZ (1987). Other species that did not transmitin our laboratory tests were A. hederae, B. helichrysi,C. vitellinae, D. noxia and M. rosae, M. dirhodum.Field-collected M. dirhodum did not transmit ZYMVor watermelon mosaic virus 2 in work by CASTLE et al.(1992).

Acknowledgements

Thanks are due to Mr F. KADAR for statistical advice.

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274 Z. Basky, T. M. Perring and I. To bia s

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Authors' addresses: Z. BASKY (corresponding author),I. To BIA S, Plant Protection Institute Hungarian Academyof Sciences 1525 Budapest P.O. Box 102 Hungary;T. M. PERRING Department of Entomology, University ofCalifornia, Riverside, CA 92521, USA

Spread and aphid transmission of ZYMV 275