BIOLOGICAL AND SEROLOGICAL CHARACTERIZATION OF VIRUSES OF SUMMER SQUASH CROPS IN YUGOSLAVIA

A survey on summer squash open field crops was carried out during 2000 and 2001 in order to identify the major viruses infecting these crops in different localities. Plants showed different types of symptoms: mild mosaic, chlorotic spotting, distinctive mosaic, blistering of leaf lamina, leaf yellowing, deformation of leaf lamina, knobbed fruits and stunting of plants. The symptoms were very variable but showed the viral nature of the investigated summer squash diseases. The collected samples were tested by bioassay and by two serological methods ELISA and EBIA using cucumber mosaic cucumovirus (CMV), zucchini yellow mosaic potyvirus (ZYMV), watermelon mosaic potyvirus 2 (WMV-2), zucchini yellow flack potyvirus (ZYFV), watermelon mosaic potyvirus 1 (WMV1), squash mosaic comovirus (SqMV) and cucurbit aphid-borne yellows polerovirus (CABYV) polyclonal antisera. In all tested samples single or mixed infection with ZYMV, CMV and WMV-2 was detected. The most prevalent virus infecting summer squash was ZYMV. This is the first report of ZYMV, the most destructive virus infecting cucurbits, in Yugoslavia. It was also proven that the identified viruses are transmissible by Aphis gossypii in a non-persistent manner, but possible role of seed in virus transmission was not confirmed.


I n t r o d u c t i o n
Cucurbit plants (cucumber, watermelon, melon and squash) are very sensitive to viral infection.Over 30 viruses have been reported to infect these plants (Z i t t e r et al., 1996).The diseases they cause can be very destructive and are not easy to be controlled.Their incidence, severity and importance varies and depends on the growth area, virus involved and the complexity of infection.The most of described viruses cause very serious losses in cucurbit production, but 6 of them have been considered to be economically the most important.They are: cucumber mosaic cucumovirus (CMV), watermelon mosaic potyvirus 2 (WMV-2), zucchini yellow mosaic potyvirus (ZYMV), watermelon mosaic potyvirus 1 (WMV-1, earlier papaya ringspot virus-PRSV) and squash mosaic comovirus Although reports from all over the world point out that viruses cause the largest number and the most destructive diseases of cucurbits, viruses of these crops have been poorly studied in Yugoslavia.No research has been done and there have been no reports on viruses infecting summer squash (Cucurbita pepo L.).A small number of previous reports in former Yugoslavia are related to viruses of watermelon (S t a k i ć and N i k o l i ć , 1966), some cultivated cucurbits (P e j č i n o v s k i , 1978), and cucumber (T o š i ć et al., 1996).
In Yugoslavia, the problem of virus infection of squash (Cucurbita pepo), attracted attention in the year 2000, with its incidence in dramatic proportion (D u k i ć et al., 2001) when a new syndrome of the disease was noticed on cucurbits which triggered etiologic investigation.

Sample collecting
The field surveys were done in 2000, in the locality of Veliko Selo, near Belgrade where the production was completely destructed and Futog, vicinity of Novi Sad and 2001 in the localities of Slanci, Begaljica, and Grocka.Samples were collected from summer squash cv.Beogradska tikvica and hybrid Ezra F1 with prominent symptoms of virus infection on leaf and small fruit with distinctive deformation, with knobby growth or deep cracks and whole habitus.Collected samples were further investigated in order to confirm the viral nature of the disease and to identify the causal agent.Investigations were performed by bioassay and serologically.

Bioassay
Pathogenicity test and biological characterization were performed by mechanical inoculation.For this purpose, tissue of young leaves or fruit with typical symptoms were homogenized in cold 0.01M phosphate buffer saline (PBS) pH 7.0 in 1:1 ratio (w/v) with abrasive added.Bioassay was performed on the following test plants:

Serological tests
In order to identify the virus, causal agent of summer squash disease, two serological methods were also used in the investigations: EBIA (Western blot) and DAS ELISA.
EBIA was performed according to the procedure of O ' D o n e l l et al. (1982) modified by H e w i s h et al. (1986).Samples were prepared as described by L a e m m l i (1970) and polyclonal antisera against CMV, ZYMV and WMV-2 (Bioreba AG, Switzerland) were used at 1:1000 dilution, where goat-antirabbit antisera (Bio-Rad Lab., Richmond CA.USA) was used at 1:2500 dilution in skimmed milk.The reaction was rated as positive if lilac blue color occurred on nitrocellulose membrane.
In DAS ELISA procedure (C l a r k and A d a m s , 1977), except the above mentioned antisera against WMV-1, SqMV, zucchini yellow flack potyvirus (ZYFV) and cucurbit aphid-borne yellows polerovirus (CABYV) were used.Commercial kits of specific polyclonal antibody and alkaline phosphataselabelled conjugate γ-globulin (Bioreba AG, Switzerland) were used at 1:1000 dilution in corresponding buffers.1g of leaf or fruit tissue was homogenized in 5 ml of 0.02M PBS with 0.05% Tween 20 and 2 % polivinyl pyrrolidone MM 40 000 (A v g e l i s and K a t i s , 1989).Substrate p-nitro phenyl phosphate (1 mg/ml) was added to plates and reactions were allowed to progress for half an hour at room temperature and absorbance values were determined with a plate reader at 405nm.Absorbance equal or greater than twofold the average for healthy control samples in the same plate -experiment were regarded as positive.

Aphid transmission
During crop examination abundant aphid colonies were recorded on diseased plants.Aphid species were identified, and simultaneously transferred to young healthy summer squash plants and kept under greenhouse conditions.Also, originating from field aphid colonies, aviruliferous adults were raised on summer squash cv.Beogradska tikvica and cabbage leaves on which they were also kept for further investigations.
Viruliferous aphid colonies from diseased field plants were transferred to individual summer squash plants where they were allowed to feed for two minutes after which plants were sprayed with insecticide.
Aviruliferous aphid adults, after a period of three hours of starving, were given a two minute acquisition period on infected summer squash plants and then aphids were transferred to healthy plants to feed for 24 hours.
Five plants of summer squash cv.Beogradska tikvica in 2-3 true leaf stage were inoculated with five adult aphids for each treatment.

Seed transmission tests
Seed transmission tests were made by using 500 seeds of summer squash cv.Beogradska tikvica and 500 seeds of hybrid Ezra F1 from commercial lot used in field production.
Seeds were sown in pots filled with steam sterilized soil and kept in greenhouse conditions.Seedlings were checked for virus by recording symptoms up to one month after emergence.In cases where virus-like symptoms occurred inoculations on Chenopodium quinoa, Cucurbita pepo cv.Beogradska tikvica and hybrid Ezra F1, serological tests by ELISA with ZYMV, WMV-2, CMV antisera and grafting by transplantation on healthy plants were done.

Field symptoms
Survey of summer squash crops in the locality of Veliko Selo showed that a large number of plots, where hybrid Ezra F1 and summer squash cv.Beogradska tikvica were grown, were completely destroyed.Infected plants demonstrated typical and severe viral disease symptoms.Also, within a given cultivar, some field plants were severely distorted and others were only mildly affected.
On leaves, fruit and in the overall growth habit of the plants symptoms were very prominent and variable, depending upon the host, the affected plant parts, and developmental stage at which the plants became infected.Plants were stunted and bushy because of shortened internodes with small, distorted or no fruit at all.Foliar symptoms ranged from mild mosaic, chlorotic spotting, distinctive mosaic, blistering of leaf lamina, leaf yellowing, through different deformation to leaf lamina narrowing.The most striking symptoms occurred on infected fruit where knobby overgrowth covered the fruit surface.Deformation and abnormal green coloration occurred on fruit.Such fruits were often small and severely distorted with proliferations or longitudinal cracks, which was especially present on fruit originating from Novi Sad.Plants infected by the time fruit set begins produced unmarketable fruit.
The same symptoms on summer squash were found in the survey of other localities (Slanci, Begaljica and Grocka) in 2001.
Described symptoms are grouped into several types (table 2.).In many cases the same infected plant showed different types of symptoms.

Test plants reaction
Samples collected during the years 2000 and 2001 have been infected with ZYMV, CMV and WMV-2, based on the reactions of test plants and host range.The reaction of test plants and the host range have shown that it is possible to differentiate mechanically transmittable viruses of summer squash (table 1).
Investigated isolates of certain viruses demonstrated no differences in host range, nor symptom type which they caused on tested plants.
CMV differs clearly from other two viruses, before all, based on the reaction of Nicotiana spp.plants (N.tabacum, N. glutinosa, N. clevelandii) where it causes mosaic, systemic type of symptoms, while ZYMV and WMV-2 are not infective for these plants.CMV is the only of three viruses infectious to Physalis floridana, Petunia hybrida, Vigna sinensis, Lycopersicon esculentum, Capsicum annuum.On Luffa cylindrica CMV causes characteristic mosaic symptoms, but WMV-2 causes latent infection.On Citrullus lanatus CMV, like ZYMV and WMV-2, causes mosaic, but in rare cases it also causes the appearance of large chlorotic local lesions.
Symptoms on test plants induced by our CMV isolates are the same as those reported by other authors (L a s t r a , 1968; C o h e n and N i t z a n y , 1963; T o b i a s and T u l i p a n , 2002).
ZYMV and WMV-2 have a very similar host range, but differ in some cases.WMV-2 opposite to ZYMV, on Tetragonia expansa, N. benthamiana and Luffa cylindrica causes latent infections which were proven by back inoculations on Chenopodium quinoa.After inoculation with WMV-2, local chlorotic lesions followed by prominent mosaic and leaf deformations appear on Chenopodium quinoa.Also, WMV-2 on Chenopodium amaranticolor inoculated leaves causes local chlorotic lesions which become necrotic in a few days, where all tested ZYMV isolates cause local chlorotic lesions which become necrotic only after the plant decays.WMV-2 isolates investigated in the course of this study cause the same symptoms on most tested plants as those previously described (P r o v v i d e n t i and S c h r o e d e r , 1970; P u r c i f u l l et al., 1984), although our isolates do not induce mosaic on Nicotiana benthamiana, but latent infection.This makes our isolates different from some described by other authors (T o š i ć et al., 1996; T o b i a s and T u l i p a n , 2002).Also, our WMV-2 isolates cause the appearance of numerous chlorotic spots on inoculated leaves of C. quinoa, as many described isolates, but they belong to a small number of isolates (L i s a and D e l l a v a l l e , 1981, P u r c i f u l l et al., 1984, T o š i ć et al., 1996) which can infect this plant systemically causing mosaic and leaf deformation.
Test plants symptoms as well as serological analysis confirmed that ZYMV, CMV and WMV-2 are present in single or in mixed infections.Out of 20 tested samples, 6 of them (30%) showed to be single virus infection, and in 9 (45%) cases the complex infections with two viruses were proven (table 2).In the remaining 5 (25%) samples complex infections with all three viruses were confirmed.ZYMV was detected in single or mixed infections in 15 (75%), CMV in 13 (65%), and WMV-2 in 9 (45%) samples.(1) ZYMV a,b,c +CMV a,b,c (1) ZYMV a,b,c +CMV a,b,c +WMV-2 a,b,c (1) CMV a +WMV-2 a,b,c (2 (2) * symptom type: 1= plant stunting; 2= mild mosaic, chlorotic spotting; 3= distinctive mosaic, leaf deformation; 4= blistering, yellowing of leaves; 5= fruit deformation, knobby overgrowth or cracks a virus identification by bioassay b virus identification by ELISA c virus identification by EBIA Symptoms induced by ZYMV, CMV i WMV-2 on cucurbits in field (table 2) are various and there is no connection between the type of symptom and the virus involved, which suggests the impossibility of diagnosing the disease based on field symptoms.

Serological analysis
Results of virus identification obtained by bioassay were confirmed by serological tests (ELISA and EBIA) (table 2).
Polyclonal antisera against ZYMV and WMV-2 could detect viruses in all tested samples by both applied serological methods used.On the other hand, CMV specific antiserum reacted positively only with four out of 13 samples containing that virus.With the remaining samples, where that virus was proven by bioassay, no serological reaction was observed.Most probably the absence of serological reaction was due to low antigen concentration or the quality of commercial CMV antisera.Polyclonal antisera specific to ZYFV, WMV-1, SqMV and CABYV did not react positively with tested samples in ELISA test.
Results show that both serological methods are sensitive and suitable for serological testing of infected summer squash plants, although ELISA has shown to be more suitable for its worldwide routine use in summer squash virus infection testing (M e n a s s a et al., 1986; Y u k i et al., 2000), and because of its possibilities of testing a large number of samples simultaneously.

Aphid transmission
Transmission of the investigated viruses were attempted with the cotton aphid Aphis gosypii which was predominant insect in surveyed fields.Some adults of Aphis gosypii, found on infected summer squash plants under field conditions, were directly transferred to summer squash test plants for inoculation.All summer squash test plants infested with viruliferous aphids developed typical symptoms and the viruses were proven by back inoculations.
In that way it was proven that investigated ZYMV, CMV and WMV-2 isolates are nonpersistently transmissable by Aphis gosypii.

Seed transmission
Out of 1000 emerged tested seeds, only two summer squash seedlings cv.Beogradska tikvica, showed local chlorotic spots on cotyledon leaves which reminded of virus caused infections.None of the applied methods as bioassay, ELISA or grafting, did not confirm the viral nature of the symptoms which occurred on these two plants.The rest of the seedlings were symptomless up to two months after emerging.
Health check of seed lots used in commercial production did not show viral infection, suggesting that the seed has no role in the epidemiology of the studied summer squash diseases.These results are in agreement with those obtained by some researchers (

C o n c l u s i o n
New disease syndrome of summer squash (Cucurbita pepo) cv.Beogradska tikvica and hybrid Ezra F1, appearing with prominent plant stunting, leaf yellowing, mosaic, blistering of leaf lamina, different leaf deformation with deformed fruit covered with knobby overgrowth, proliferations or longitudinal cracks occurred for the first time in the year of 2000, and repeated in 2001.In some localities the incidence of this disease completely destroyed or seriously endangered summer squash crops.Infected plants demonstrated typical and severe viral disease symptoms.
Etiologic investigations have shown that the disease is of a viral nature.Identification of the virus was done using bioassay and serological analysis.Biological characterization of the viruses was done on different indicator plants, in order to determine the host range and plants reaction.Samples were also tested by ELISA using polyclonal antisera against CMV (cucumber mosaic cucumovirus), ZYMV (zucchini yellow mosaic potyvirus), WMV-2 (watermelon mosaic potyvirus 2), ZYFV (zucchini yellow flack potyvirus), WMV-1 (watermelon mosaic potyvirus 1), SqMV (squash mosaic comovirus) and CABYV (cucurbit aphid-borne yellows polerovirus).Serological investigations by EBIA method were done using polyclonal antisera against CMV, ZYMV and WMV-2.
Based on symptomatology of test plants and confirmed by serological reactions, it is concluded that the investigated samples have been infected in single or mixed infections with ZYMV, CMV and WMV-2.ZYMV, which was reported for the first time in Yugoslavia, was identified in most samples.
Aphid colonies found on summer squash plants under field conditions were determined as Aphis gossypii.Transmission tests have shown that identified viruses are transmitted by that vector in a non-persistent manner.
Checking infectivity in seed lots used in commercial production of summer squash, did not show the possible role of seed in disease transmission.
(SqMV) (W e b b et al., 1965; M i l n e et al., 1969; L o v i s o l o , 1980; N a m e t h et al., 1986; K y r i a k o p o u l o u and V a r v e r i , 1991; D i k o v a , 1995; L i s a et al., 1997; G a l l i t e l l i and D a v i n o , 1998; T o b i a s and T u l i p a n , 2002).

T a b . 2 .
-Viruses identified and their connection with symptom types on infected plants in the R o b i n s o n et al., 1993; O r o z c o -S a n t o s et al., 1994), while some researchers (S c h r i j n w e r k e r s et al.,1991) pointed out transmissibility of ZYMV with summer squash seed, but in very low percentage.T o b i a s and T u l i p a n (2002) also point out that seed transmission of ZYMV can be achieved in a few promiles resulting in 7-8 infected plants per hectare, which provides a source of inoculum.Neither for WMV-2 (S t a k i ć i N i k o l i ć , 1966) nor for CMV (Z i t t e r et al., 1996) seed transmission has been shown.
Luffa acutangula, and that isolate is different from ours in its infectivity to Nicotiana benthamiana.