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Introduction
There are three economically important Cucurbita species, namely C. pepo (zucchini), C. maxima (squash), and C. moschata (pumpkin), which have different climatic adaptations and are widely distributed in agricultural regions worldwide (Paris et al., 2005). Zucchini, winter squash and pumpkin are three of the most important cucurbit vegetable crops in Turkey. The annual winter squash and pumpkin production in Turkey was 93 672 t in 2014. Samsun city, located at the central Black Sea region, has a 10.5 % share of Turkey production with 9 913 t (Turkstat, 2014).
Cucurbit species are susceptible to diseases that attack the roots, foliage, and fruit (Howard et al., 1994) and about 39 different virus species cause cucurbit diseases (Ali et al., 2012). Zucchini yellow mosaic virus (ZYMV), a common viral pathogen, reduces fruit quality and yield (Bonilha et al., 2009), and it is a potyvirus with flexuous, filamentous particles of single-stranded RNA, about 750 nm long, and one coat protein (Spadotti et al., 2015). This virus is easily transmissible to a fairly wide range of hosts and is transmitted by 26 species of aphids in a non-persistent manner (Katis et al., 2006).
There are few studies about the characteristics of the disease occurrence and the pathogenicity of the ZYMV on summer and winter squashes in Turkey, it is widely distributed in the country regions and has biological variability that causes devastating epidemics in a range of cucurbit crops. Therefore, the objective of this study was to determine occurrence, prevalence and biological characteristics of ZYMV on different squashes in major cucurbit producing areas of the Black Sea region of Turkey.
Materials and Methods
Surveys
The most reported areas for high production of cucurbit crops in Samsun province were subjected to survey of infected plants with ZYMV. The surveyed plants were zucchini, winter squash and pumpkin. Symptomatic leaf and fruit samples were collected from districts: Bafra (28), Çarşamba (60), Tekkekoy (90), Terme (25), and Canik (22) (Table 1). The symptoms observed on the leaves, and fruit were recorded at the time of sample collection (Jossey and Babadoost, 2008). Collections were made from 2015 to 2016 during summer (70 samples) and winter seasons (155 samples) from each site. The collected samples were placed in plastic bags, returned to the laboratory on the same day and kept at 4 ºC until used.
Serological identification
A polyclonal antiserum kits for ZYMV (Bioreba) and Polystyrene 96 well plates (TPP) were used in this study. The double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) was used for the examination of samples. Samples for DAS-ELISA were prepared by grinding of leaf tissue in phosphate buffered saline, pH 7.4 with 2 % PVP and 0.2 % of bovine albumin (1:5 ratio). Plates were incubated during 2 h at 24 °C after pipetting the substrate buffer, and the absorbance values were read at 405 nm using a microplate reader and also confirmed visually after incubation for 2 h at room temperature. All samples tested in two replicate wells and with an absorbance value greater than three times that of a negative control, were rated as positive (Spadotti et al., 2015).
Mechanical inoculation
Mechanical inoculation was performed on carborandum dusted cotyledon leaves at cotyledonary leaf stage. All inoculations were repeated three times. Six plants of each of cucurbit species were used for each treatment. Sap prepared from ELISA-positive zucchini (ZYMV-CA), squash (ZYMV-TE) and pumpkin (ZYMV-BA) samples and C. pepo, C. maxima, C. moschata, Cucumis sativus, and Citrillus vulgaris plants, using 0.01 M phosphate buffer (pH 7.0), was mechanically inoculated onto carborundum-dusted plants of each Cucurbit test plant (Hosseini et al., 2007). Control plants were buffer inoculated. The leaves were then rinsed with water, and plants were maintained in a controlled climate room at 24 ºC and 90 % relative humidity for observation. The inoculated plants were tested for ZYMV infections 30 d post inoculation using DAS-ELISA
Results and Discussion
The virus-like disease symptoms most commonly observed in the fields were mosaic, and mottling of the leaves and fruit malformations with heavy yield losses. The virus disease incidence assessed by a visual search for virus-like symptoms was 37 % of cucurbit fields surveyed. A total of 225 symptomatic samples were examined from five different regions. DAS-ELISA results showed that 84 samples of zucchini, winter squash and pumpkin out of 225 cucurbit samples tested reacted positively with ZYMV. In C. pepo, C. maxima, and C. moschata, ZYMV was prevalent virus and was detected in 34 of 70, 28 of 90, and 22 of 65 samples, respectively (Table 1).
ZYMV was found in all the cucurbit growing regions surveyed. The highest virus incidence among the surveyed regions was recorded in Carsamba (56.6 %), followed by Bafra (35.7 %), Terme (32.0 %), Tekkekoy (31.1 %), and Canik (18.1 %) (Table 2).
Table 2 Occurrence of ZYMV in squash samples collected in 2015-2016.
| Regions | No. of fields surveyed | Samples tested/ infected | ZYMV | % | |
| 2015 | 2016 | ||||
| Bafra | 10 | 15/6 | 13/4 | 10 | 35.7 |
| Carsamba | 20 | 30/18 | 30/16 | 34 | 56.6 |
| Tekkekoy | 30 | 45/12 | 45/16 | 28 | 31.1 |
| Terme | 8 | 13/5 | 12/3 | 8 | 32.0 |
| Canik | 7 | 12/3 | 10/1 | 4 | 18.1 |
| Total | 75 | 115/44 | 110/40 | 225/84 | 37.3 |
ZYMV isolates (CA, TE, and BA) were mechanically inoculated in test plants for biological characterizations and symptom development was observed after 7 d inoculation. Reactions of tested cucurbit plants are summarized in Table 3.
Table 3 Symptoms caused by ZYMV on test plants and ELISA positive samples.
| Species | Symptoms*/ DAS-ELISA** | ||
| ZYMV-CA | ZYMV-TE | ZYMV-BA | |
| C. pepo | Mo, LM */ +** | Mo / + | Mo, LM / + |
| C. maxima | Mo, LD / + | Mo, LD / + | Mo / + |
| C. moschata | Mo / + | Mo / + | Mo / + |
| C. sativus | Mo / + | Mo / + | Mo / + |
| C. vulgaris | NS / - | NLL / + | NS / - |
* Mo: mosaic, LM: leaf malformation, LD: leaf deformation, NLL: necrotic local lesion, and NS: non-symptoms. ** +: Virus detected in DAS-ELISA.
All ZYMV isolates systemically infected C. pepo, C. maxima, C. moschata, and C. sativus. In contrast, ZYMV isolated from winter squash produced necrotic local lesions on C. vulgaris (Table 3). More than 39 different viruses are reported from the family Cucurbitaceae, but ZYMV is one of the most economically important viral diseases of this family (Bonilha et al., 2009). This virus was first reported in Northern Italy by Lisa et al. (1981), and it is responsible for significant loses in yield and quality of cucurbitaceous vegetable worldwide (Spadotti et al., 2015).
In our study, 225 symptomatic leaf samples of cucurbits were collected in 2015-2016 and tested for the presence of ZYMV. Data showed that ZYMV was very common in the research areas (Table 1) and infected plants were found in all cultivated squashes assayed including zucchini, squash, and pumpkin. Yakoubi et al. (2008) studied population structure of ZYMV in Tunisia and DAS-ELISA tests showed that 92 samples of zucchini and squash out of 106 cucurbit samples tested reacted positively with a ZYMV polyclonal antiserum, which was found in all the cucurbit growing regions surveyed: Bizerte (54 positive samples out of 55 tested, Cap Bon (29/37) and Monastir (9/14).
In our study, the occurrence of ZYMV was 48.5, 33.8, and 31.1 % in zucchini, pumpkin, and squash, respectively (Table 1). Surveys conducted to identify viruses infecting pumpkin and squash in Illinois, USA, detected ZYMV in 18, 4, and 4 % of the samples tested in 2004, 2005, and 2006, respectively (Jossey and Babadoost, 2008). According to Prendeville et al. (2012), spread degree of the virus varies greatly among regions and years. In our study, incidence of ZYMV was 38.2 and 36.3 % in 2015 and 2016, respectively, and it was recorded at different commercial open-fields in five regions of Samsun (Table 2).
ZYMV-infected plants exhibit severe leaf mosaic, yellowing and eventually shoestring symptoms in the leaves. The fruits are stunted, twisted and deformed, resulting in reduced yield and the plants unmarketable; especially zucchini squash (Spadotti et al., 2015). In our study, typical ZYMV infected symptoms of leaves mosaic and mottling, and fruit malformations were observed from summer and winter squashes naturally infected with ZYMV, which agrees with results reported by Lecoq et al. (2009).
For biological characterizations of ZYMV, isolates were mechanically inoculated onto cucurbit plants and symptom development was observed after 7 d inoculation. Watermelon plants exhibited necrotic spots 1 week post-inoculation, whereas mosaic with leaf deformation were observed in all inoculated zucchini, winter squash, pumpkin, and cucumber plants 7 d post-inoculation; besides, there was biological variability among isolates. Because of its aggressiveness and virulence, ZYMV is a serious threat to cucurbit production worldwide (Singh et al., 2003).
In our study ZYMV-CA was more aggressive in cucurbits, but TE and BA isolates could not infect watermelon plants, induced milder symptoms in winter squash, pumpkin, and cucumber plants. Similarly, ZYMV isolates were analyzed in 11 isolates sampled from cucumber, squash and zucchini between 2001 and 2006 in Slovakia and Czech Republic; most of isolates showed typical mosaic symptoms on zucchini genotypes, and SE04T isolate was unique in failing to produce symptoms, although virus presence was detected by DAS-ELISA in all tested cucurbit genotypes (Glasa et al., 2007).
In aphid transmission under natural conditions, the emergence and spread of aggressive variants might be enhanced by aphid preference for symptom-expressing plants (Fereres et al., 1999). The virus detected in our study was spread efficiently by aphids, seeds, and mechanical inoculation (Yakoubi et al., 2008). ZYMV is transmissible by aphids in a non-persistent manner, that is, rapid transmission in several minutes without any long-time acquisition and inoculation feeding. Our results confirmed the presence of ZYMV in the Middle Black Sea region and showed that the virus is widespread in Cucurbit species. The virus was detected in all zucchini, winter squash, and pumpkin fields, but there were higher incidences of virus infection in some regions, as compared to others. It seems that in an area, presence of an inoculum source, especially infected seeds, was more important for incidence of virus infection than location of fields (Golnaraghi et al., 2004).
Conclusions
ZYMV is spread in cucurbits and most of the commonly grown squash cultivars are infected. Widespread occurrence of ZYMV in major summer and winter squash growing areas of the Black Sea region of Turkey would implicate its higher impact on cucurbit production throughout the country. We report biological characteristics of ZYMV isolates from cucurbits, which might provide better understanding of ZYMV epidemiology.
