Infection cucurbit fields to most important plant viruses and phylogenetic analysis

Viral diseases are one of the limiting factors for cucurbit production around the world that some of these viruses can cause a severe reduction in crop yield. In the present study, 92 leaf and fruit samples of various species of cucurbits were collected from different farms in Zanjan and Hamedan provinces, based on descripted symptoms such as yellowing, mosaic, deformation, leaf asymmetry, and wrinkling of leaf veins, to detect and investigate the incidence of some important viruses in cucurbit fields. After total RNA extraction, RT-PCR were conducted using specific primers for Cucumber mosaic virus (CMV), Cucurbit aphid-borne yellows virus (CABYV), Cucumber green mottle mosaic virus (CGMMV), Squash mosaic virus (SqMV), Ourmia melon virus (OuMV), Zucchini yellow mosaic virus (ZYMV), and general primers of the Orthotospovirus genus to amplify a part of the genome corresponded with each of these viruses. The results showed evidence of the presence of CMV, CGMMV, ZYMV, and CABYV, in the tested samples, while OuMV, SqMV and Orthotospoviruses were not detected in any of the tested samples. Among the detected viruses, CABYV was detected as the most common virus. Phylogenetic analysis of the new isolates and other isolates from different countries showed that CMV strains were placed in both subgroups I and II. The detected ZYMV strains from Zanjan were clustered with the Vietnamese , and CGMMV strains were grouped with strains from India, Pakistan, Uzbekistan, the Netherlands, and Spain in the cluster II. CABYV strains were grouped in the Mediterranean, Asian, and a new independent group, along with the Taiwanese and Indonesian. In addition, mixed infections of those virus infections were detected commonly in squash and cucumber. Especially, CGMMV+CABYV, CMV+CABYV and the infection of ZYMV+CABYV+CMV, ZYMV+CGMMV+CMV, and ZYMV+CGMMV+CABYV mixed infections were detected. This results will be useful in developing management strategies to manage of cucurbit viruses.
Introduction
The Cucurbitaceae family, after Solanaceae, ranks second in terms of economic importance among horticultural plant species worldwide (Renner and Schaefer., 2016). Cucurbits comprise over 800 species of plants in 120 genera (Welbaum, 2015). According to the statistics of the Food and Agriculture Organization (FAO), global production of cucurbits was reported to be around 1,205,679 tons in 2020. Viral diseases are important factors for the limiting of cucurbit production worldwide. More than 70 species of plant viruses have been reported to infect cucurbits around the world (Lecoq and Katis., 2014). Plant viruses are responsible for more than $30 billion in annual losses in crops (Sastry et al., 2019). Cucumber mosaic virus (CMV), Cucurbit aphid-borne yellows virus (CABYV), Papaya ringspot virus (PRSV), Zucchini yellow mosaic virus (ZYMV), and Watermelon mosaic virus (WMV) are viruses that are widely spread on cucurbits in various regions, especially in the Mediterranean area. These viruses mostly cause mosaic, yellowing, and deformation on leaves and malformation of fruits (Lecoq and Desbiez., 2012). In Syria, as one of the major areas of cucurbit production, ZYMV, WMV, CMV, CABYV, and Pepo aphid-borne yellows virus (PABYV) have been identified using molecular methods (Chikh-Ali et al., 2019). A recent study in northern and central Argentina showed high levels of WMV incidence in several species of cucurbits (Pozzi et al., 2020). A general survey in 2017 and 2018 showed that WMV is the most common and widespread virus observed in all regions of cucurbit growth in Argentina, with a relative incidence of 46%, followed by 24% for ZYMV and 20% for Papaya ringspot virus. Additionally, CMV and Squash mosaic virus (SqMV) have been reported on cucurbits in different regions of Azerbaijan (Huseynova et al., 2017; Verdin et al., 2018). Several viruses, including WMV, CABYV, SqMV, CMV, CGMMV, ZYMV, Cucumber yellow stunting disorder virus (CYSDV), and OuMV, have been reported in cucurbit plants in Iran (Mehrvar and Zakiaghl, 2021 Vafaei, and Mahmoodi; 2017, Gerami Nooghabi et al., 2022, Salehzadeh., 2018, Mohammadi et al., 2016, Maghamnia et al., 2018, Keshavarz and Izadpanah, 2004, Gholamalizadeh et al., 2008). ZYMV and CABYV are among the most common viruses infecting cucurbit plants in Iran and have been reported in several cultivation areas (Bananej et al. 2008; Massumi et al. 2011; Mohammadi et al. 2016; Sokhandan-Bashir et al. 2013; Abou Jawdah et al.,2000; Kassem et al., 2007). A survey of the prevalence of CMV, ZYMV, CGMMV, CYSDV, and Cucurbit chlorotic yellows virus (CCYV) in cucurbit cultivation areas of Lorestan Province showed that cucurbit plants are infected with all tested viruses, and reports suggest that they are present in most cucumber cultivation areas in Iran (Hasanvand and Shams-bakhsh., 2017). CMV, ZYMV, CGMMV, and WMV have been traced in cucumber, pumpkin, and watermelon fields in the northwest and west of Iran using RT-PCR method (Mohammadi et al., 2016). The simultaneous infection of two or more viruses in plants is a natural occurrence and may have an impact on the evolution and epidemics of viruses (Edwardson, 1966). In some cases, mixed infection of two unrelated viruses may exacerbate symptoms, and it may affect the replication of the infecting viruses (Hull, 2013). The effect of Cucumber mosaic virus and Tomato mosaic virus (ToMV) on the growth and performance of tomato plants has been observed. The combination of ToMV + TYLCV + CMV and ToMV + TYLCV in mixed infection shows greater synergistic effects on disease symptoms, while a combination of ToMV + CMV has caused milder symptoms (Mohamed, 2010). The combination of CMV and WMV with ZYMV, Papaya ringspot virus (PRSV) and CGMMV led to a decrease in symptoms. The dual mixed of ZYMV+CMV and ZYMV+PRSV-W is a common combination of viruses found in infected pumpkin samples (Nontajak et al., 2014). PRSV and ZYMV create various symptoms such as mosaic, stunted growth, distortion of stems and leaves, and shoestring-like tendrils (Fletch et al., 2000). The majority of viruses that infect cucurbits are widespread in different geographic regions of the world, and their host range is not limited to one plant species. Accurate diagnosis of pathogens is necessary for plant disease management, and the identification and detecting of viruses are crucial to conduct further research to prevent and control these viruses in the future. The economic value and widespread consumption of products derived from Cucurbitaceae plants make them highly significant. However, viral infections pose a significant threat to these plants, causing estimated losses of 3 to 5 percent of total vegetable production, with the potential for even higher losses in some cases (Caciagli, 2010). So far, no comprehensive information is available on the viruses infecting Cucurbitaceae plants in Zanjan and Hamedan provinces, and this research aims to investigate the level of infection of some Cucurbitaceae farms with important viruses, and to examine the level of mixed virus infection in collected plant samples from the Cucurbitaceae family in different regions of Zanjan and Hamedan