FIRST REPORT OF Cucumis melo endornavirus WITH A NEW HOST, THE GHERKIN (Cucumis anguria Linn.), IN TURKEY

: More than 50 viruses causing infection in members of the family Cucurbitaceae have been identified in the world so far. Because of the development of virus detection methods, new viruses are added to the known infectious cases list every day. One of the viruses recently identified is the Cucumis melo endornavirus (CmEV) which has been reported from different countries all over the world. However, no study for determination of CmEV has been done so far in Turkey. For the purpose of this study, 59 cucurbit plants showing virus and virus-like symptoms were collected from Manisa and İzmir provinces in Turkey. The samples were tested, for presence of CmEV, by reverse-transcriptase polymerase chain


Introduction
Turkey is characterized with a wide range of ecological diversity which allows cultivation of different plant taxa one of which includes the cucurbit plants in the family Cucurbitacea (Çat et al. 2016). The currently known 825 species of this family are placed in 118 genera (Jeffrey 1980). The most important species of this family include melon (Cucumis melo Linn.), watermelon (Citrullus lanatus Thumb.), gherkin (Cucumis anguria Linn.), and zucchini/squash/pumpkin (Cucurbita spp.) all which can be cultivated in almost every region of the world and provide important economic inputs to their producers.
There are many viral diseases known to affect members of this important plant family (Zitter et al. 1996) by causing economic losses. New viruses are increasingly being added to the list of disease agents and they threaten the production of cucurbit plants. Some of these viruses are routinely studied, while others are not. Therefore, there is no reliable available information about the importance and prevalence of these viruses.  (Fukuhara et al. 2006). CmEV sequences from South Korea are available in the GenBank. However, there has been no attempt in Turkey so far to detect the presence of CmEV infection. In the present study, field studies were performed on cucurbit plants grown in Manisa and İzmir provinces on Turkey and the presence of the CmEV as the causal agent was investigated.

Sampling and virus detection
Samples were collected in randomly selected fields in İzmir and Manisa provinces among cucurbit plants only showing the virus and virus-like symptoms (Fig. 1). The field studies were carried out from June to September in 2019. When similar symptoms were observed in the same field, only three samples were collected.
The presence of CmEV in the collected samples was determined by reverse-transcriptase polymerase chain reaction (RT-PCR) using a virus-specific primer pair. Before performing RT-PCR, total nucleic acid (TNA) isolation was performed by the cetyl trimethylammonium bromide (CTAB) method (Li et al. 2008). The resulting TNAs were checked by agarose gel electrophoresis and stored at -80 ºC until used.
To determine the presence of CmEV in the resulting TNAs, complementary DNAs (cDNAs) were initially synthesized using the RevertAid First Strand cDNA Synthesis Kit (Thermo Scientific ™, USA). The presence of CmEV was then screened by PCR with the CmEVprimer pair (Table 1)

Sequence analysis
Five samples were chosen among the samples which were accepted to be infected with CmEV based on host species and the geographic origin, where they were obtained from. The resulting RT-PCR products were purified by EZ-10 Spin Column PCR Products (BioBasic, Canada) and sequenced bilaterally.
The assembled sequences were used to match with other world CmEV isolates from the GenBank (

Results
A total of 59 cucurbit plants with viruses and viruslike symptoms were collected. Forty-nine of these were melon, seven were pumpkins, and three were gherkins. CmEV was detected in 47 of the samples (Table 3).
With the exception of five collected melon samples, nearly all were found to be infected with CmEV. Three CmEV infections were detected in gherkin plants. CmEV infection was detected in none of the pumpkin plants.
As a result of the sequence analysis performed, it was determined that Turkish CmEV isolates showed 93%-99% and 94%-99% identities with each other at the nucleotide and amino acid levels, respectively. In multiple sequence alignments with world CmEV isolates, it was observed that Turkish CmEV isolates showed 93%-98% and 91%-98% identities at the nucleotide and amino acids levels, respectively. The highest nucleotide identity rate between the Turkish and world CmEV isolates was found to be 98% between MAN25 with BRA/TO-74/2010 and BRA/TO-23/2014 isolates, while the least nucleotide identity was found to be 93% between MAN22 with CL01 and SJ1 isolates (Fig. 2).
Based on the phylogenetic analysis, all of the Turkish CmEV isolates, except MAN25 and IZM36, were closely related and formed a separate clade. While the world isolates form a clade with MAN25, IZM36 was found as a separate clade that consists of only itself (Fig. 3).   It is known that endornaviruses infect plants and fungi. There are many economically important crops that are infected with these viruses. Some of these crops are rice, barley, pepper, and common bean (Wakarchuk & Hamilton 1985, Zabalgogeazcoa & Gildow 1992, Fukuhara et al. 1993, Okada et al. 2011). In a recent study from the United States (US), endornaviruses were detected in non-cultivated plant species (Herschlag et al. 2019). In addition to the known host range of endornaviruses, gherkin was found to be a new host for CmEV as the result of this study.
In a study carried out in the US, it was reported that CmEV populations had 10% and 6% genetic variation with each other at the nucleotide and amino acid levels, respectively (Sabanadzovic et al. 2016). In this context, the identity rates obtained in this study showed great parallelism with that study.
It was determined that the isolates did not show phylogenetic distribution according to their geographic origin and host (Fig. 3). However, it is considered necessary to perform analyses using a much larger number of isolates with larger sequences for more reliable results.
As a result of the recent worldwide studies, the host range of endornaviruses has been expanded. However, the impact of these viruses on crop yields or growth parameters is still unclear (Escalante et al. 2016, Fukuhara 2019, especially in cases in which the CmEV infected plant is co-infected with severe viruses. To the best of our knowledge, this is the first report of CmEV with gherkin (C. anguria Linn.) as a new host both in Turkey and worldwide. Moreover, it is thought that the virus may present a more extensive worldwide distribution than previously reported.