Introduction

Turkey is one of the few countries in the world that possesses all the necessary climatic conditions for the most effective conduct of viticulture activities. The grapevine (Vitis vinifera L.) is one of the temperate zone plants that can grow between the 30 °C north and south latitudes worldwide, and both the climate and geographical structure of our country are highly suitable for viticulture. As a result, viticultural activities have been carried out in our country for many years, both in traditional and modern ways (Şen and Güler 2012).

V. vinifera species dominate Turkey’s viticulture industry, including popular grape varieties such as ‘Merlot’, ‘Cabernet Sauvignon’ and ‘Chardonnay’. Wine growing in Turkey dates back to around 8000 BC, as evidenced by archaeological finds, and continued into ancient times. Ancient Anatolian peoples such as the Hittites were known for viticulture, and the quality of Turkish wines was recognised by the ancient Greeks and Romans. During the Ottoman Empire, wine production became a major industry with numerous vineyards and wineries established throughout the country. Turkey has a rich history of viticulture. Archaeological evidence suggests that grapes were grown in the region as early as 8000 years ago. Since then, over 1200 different grape varieties have been grown in Turkey, making it one of the most diverse grape-growing regions in the world (Bölek and Güler 2018).

Like all plants, viticulture is influenced by numerous factors that affect quality and productivity, including pathological and environmental factors. Grapevines are particularly susceptible to various viruses and other virus-like agents. Approximately 60 viruses are found in grapevines worldwide (Goszczynski et al. 2017; Zhang et al. 2019). V. vinifera viruses can exhibit a variety of symptoms in plants and can sometimes be fatal (Martelli 2010). These symptoms may include yellowing or wilting of leaves, leaf drop, reduced leaf size, flower drop, and small, irregular fruit shapes. Additionally, the effects of some viruses can negatively affect plant growth and development. Moreover, V. vinifera viruses can weaken the plant’s immune system, making it more susceptible to attacks by other pathogens (Martelli et al. 2007).

Furthermore, V. vinifera can adversely affect fruit production. Some viruses can lead to a decrease in fruit production and quality of harvested fruits. They can also cause physical damage to fruits such as spots, cracks, and various deformations. The diagnosis of V. vinifera viruses is typically established through laboratory tests on leaf or stem samples from plants. These tests include methods such as enzyme-linked immunosorbent assay (ELISA), polymerase chain reaction (PCR), and Western blot (Al Rwahnih et al. 2013). Multiplex PCR enables the simultaneous amplification and detection of multiple target sequences in the same reaction. Using multiplex PCR, the related viruses were detected and differentiated in the same sample. This approach significantly improves the efficiency and cost-effectiveness of the detection process by reducing the number of required reactions, resulting in time and resource savings. The use of multiplex PCR in this study provides several advantages.

First, it allows the detection of multiple grapevine viruses in a single reaction, enabling a comprehensive assessment of viral infections in the tested samples. Second, multiplex PCR enhances the sensitivity of the detection method, improving researchers’ capabilities to identify low levels of viral presence. Finally, this technique facilitates the differentiation of closely related viruses, underscoring its importance in accurate disease diagnosis and appropriate management strategies.

This study emphasizes the significance of multiplex PCR in grapevine virus identification, highlighting its speed, cost-effectiveness, enhanced sensitivity, and accurate pathogen characterization. Traditional methods, such as serological analyses and biological indexing, often lack the ability to simultaneously detect multiple viral pathogens, making them time-consuming and labor-intensive. In contrast, multiplex PCR using specific primer sets enables simultaneous amplification of multiple target DNA sequences, increasing efficiency and reducing analysis time. Additionally, this technique increases sensitivity by amplifying multiple target regions, leading to improved virus detection. The cost-effectiveness of multiplex PCR arises from its ability to optimize resource utilization by detecting multiple viruses in a single reaction. Multiplex PCR streamlines the diagnostic process and facilitates the identification and management of grapevine viruses.

Materials and Methods

The primary materials for the study consisted of grapevine plants showing virus symptoms in vineyards located in the Turkish provinces of Manisa, Izmir, and Denizli (a total of 320 plants). These materials included molecular chemical kits used for the diagnosis of grapevine viruses, primers, and the necessary chemical substances for molecular analysis. Grapevine plants displaying virus-like symptoms (mosaic, colour breaking in veins, ring spots, vein banding, flattening of shoots, cracking, shape deformity, stunted growth) were collected from 1‑year-old shoots, including leaf and shoot samples. Samples were also collected from plants showing reddening and downward curling of leaves, especially in lower leaves (Fig. 1).

Fig. 1
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Virus symptoms observed in vineyard samples: a, b vein banding on leaves, c yellowing and deformation of leaves, d color change in leaves, e deformation in fruits, f deterioration in general appearance, g brown spots in veins

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Primer Design

Primer sequences were generated by inputting specific criteria and characteristics into the design process, utilising information available in the gene bank. The primers were designed to ensure that they did not interact with each other in the reaction, allowing for the simultaneous detection of multiple agents in a multiplex format. To achieve this, various computer programs and manual calculations have been employed to control primer design with regard to features such as self-dimer and heterodimer formation.

In multiplex PCR, primers were designed to have similar annealing temperatures to function under the same conditions (Gambino et al. 2006; Morelli et al. 2020). In this context, criteria such as primer length, primer specificity, secondary structures (dimer formation, hairpin formation, etc.), GC content, base sequence arrangement, primer melting and annealing temperatures, 3’ end stability, and G/C clamp were considered when designing the multiplex PCR primers (Figs. 2, 3, 4, 5 and 6).

Fig. 2
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Graphical representation of the primer designed for grapevine fanleaf virus (GFLV) on the genome

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Fig. 3
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Graphical representation of the primer designed for grapevine fleck virus (GFkV) on the genome

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Fig. 4
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Graphical representation of the primer designed for grapevine leafroll-associated virus (GLRaV) on the genome

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Fig. 5
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Graphical representation of the primer designed for grapevine virus A (GVA) on the genome

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Fig. 6
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Graphical representation of the primer designed for arabis mosaic virus (ArMV) on the genome

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Verification of Primers Designed Using the Blast Method

Primers designed by considering specific criteria and utilizing the NCBI GenBank data were individually subjected to BLAST analysis. This analysis aimed to verify their alignment with genomes and viruses in the GenBank and determine the percentage of similarity to specific organisms.

Molecular Methods

Total nucleic acid (TNA); Nucleic acid extraction from the plant samples was conducted using the total nucleic acid (TNA) extraction method and Qiagen RNA extraction kit protocols. The samples were stored in a deep freezer at −80 °C until cDNA synthesis and PCR were performed (Foissac et al. 2001; Ekren et al. 2023).

Complementary DNA (cDNA) synthesis was performed on samples that had undergone TNA extraction (Smith et al. 2018). For this purpose, a cDNA synthesis kit (Thermo Scientific) was used following the manufacturer’s instructions (Atik and Paylan 2023; Paylan et al. 2014; Kusznerczuk et al. 2019).

The primers used for grapevine fanleaf virus (GFLV), grapevine fleck virus (GFkV), grapevine leafroll-associated virus 1 (GLRaV-1), arabis mosaic virus (ArMV), and grapevine virus A (GVA) in multiplex PCR studies are listed in Table 1. The PCR process was carried out in a total volume of 50 μl according to the procedure recommended by Thermo Scientific (Tang et al. 2020). In sterile PCR tubes, 25 μl of 2 × PCR master mix, 1 μl of primer 1, 1 μl of primer 2, 2 μl of cDNA, and 21 μl of nuclease-free water were added. The tubes were placed in a PCR machine and a specific program for the virus to be tested was applied.

Table 1 Primer sequences, polymerase chain reaction product size, sequence position in the viral genome, and characteristics
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Throughout the procedure, gloves were worn, and a tube containing only the mixture (water control) was subjected to the amplification process in the PCR program to identify any possibility of contamination. Agarose gel electrophoresis was performed in 100 ml of 1X TAE buffer containing 1.5 g of agarose and electrophoresis was conducted at 100 V on a horizontal apparatus for 60 min in 1X TAE buffer solution.

Research Results

Results of Primer Design and Blast Studies

In this study, the characteristics of the primers designed for GFLV, GFkV, GLRaV‑1, ArMV and GVA agents were determined and the BLAST results of these primers were identified. For each agent, the description, NCBI code, molecular type (nucleic acid), query coverage length, distance tree results, percentage similarity rates, and accession numbers were determined (Figs. 7, 8 and 9).

Fig. 7
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Multiple sequence alignment results for grapevine fanleaf virus (GFLV) primer

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Fig. 8
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Multiple sequence alignment results for arabis mosaic virus (ArMV) primer

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Fig. 9
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Multiple sequence alignment results for grapevine virus A (GVA) primer

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Comparisons with other agents in the NCBI database were performed for the five viral agents in this study to draw similarity rates and phylogenetic trees. A graphical representation (blast tree view) was made of which isolates in the world were associated with the molecular structure and isolates (raw data 1).

The Multiple Sequence Alignment Viewer program version 1.22.2 was used to determine sequence similarities and differences between the primers designed for GFLV, GFkV, GLRaV‑1, ArMV, GVA, and other NCBI-matched isolates, as well as the start and end points in the genome region, by comparing the sequence IDs and start and end genomes of the organisms.

Findings of Multiplex PCR Studies

In the context of multiplex PCR studies, the presence of the aforementioned viruses, GFLV, GFkV, GLRaV‑1, ArMV, and GVA, was determined in a total of 320 plant samples (Fig. 10).

Fig. 10
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Gel results for grapevine fanleaf virus (GFLV), grapevine virus A (GVA), grapevine leafroll-associated virus‑1 (GLRaV‑1), grapevine fleck virus (GFkV), and arabis mosaic virus (ArMV) as determined within different bp ranges in the context of multiplex PCR analysis; GFLV 650 bp, GVA 500 bp, GLRaV‑1 310 bp, GFkV 280 bp, and ArMV 110 bp

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In the analyses carried out on samples from different districts of Manisa, the following numbers of infected samples were observed: 20 samples from Ahmetli, 13 infected; 55 samples from Alaşehir, 26 infected; 45 samples from Salihli, 13 infected; 40 samples from Sarıgöl, 12 infected; and 35 samples from Turgutlu, nine infected. In the analysis of samples collected from Izmir district, the number of infected samples was as follows: 30 samples from Menderes, 13 infected; 30 samples from Menemen, 15 infected; and 30 samples from Urla, eight infected. From the Çal district of Denizli, 35 samples were collected and 10 infected samples were identified (Fig. 11).

Fig. 11
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Distribution of viral agents determined by province and district. GFLV Grapevine fanleaf virus, GLRaV‑1 Grapevine leafroll-associated virus‑1, GFkV Grapevine fleck virus, GVA Grapevine virus A, ArMV Arabis mosaic virus

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Of the 119 infected samples, 28 were identified as GFLV, 31 as GLRaV‑1, 12 as GFkV, 21 as GVA, and 27 as ArMV. The overall infection rate in the 320 tested samples was 37.1%, with GFLV at 8.75%, GLRav‑1 at 9.6%, GFkV at 3.7%, GVA at 6.5%, and ArMV at 8.4%.

Conclusion and Discussion

Sabanadzovic et al. (2009) analyzed a collection of 11 grapevine varieties for the presence of known and novel viruses. Among these pathogens, 29 infectious viruses with significant similarities to known plant viruses were identified. In this study, they also detected grapevine fanleaf virus (GFLV), grapevine leafroll-associated virus 2, and grapevine virus A (GVA) and characterized new viruses by sequencing their full-length genomes and analyzing their phylogenetic relationships with other known viruses. They also developed specific reverse transcription-polymerase chain reaction (RT-PCR) assays to detect new viruses in grapevine samples. The identification of new viruses in grapevine samples highlights the importance of continuous monitoring and surveillance of grapevine viruses. The development of specific RT-PCR experiments for new viruses provides a valuable tool for the early detection and management of these viruses in grapevine production systems (Cretazzo et al. 2017; Yang et al. 2021).

The first grapevine viruses detected using multiplex PCR were reported in the early 2000s. Karasev and Nikolaeva (2002) published a study describing the use of multiplex PCR for the detection of five grapevine viruses, including GFLV, arabis mosaic virus (ArMV), tomato black ring virus (TBRV), GVA, and grapevine virus B (GVB). This study demonstrated the high specificity and sensitivity of multiplex PCR for the detection of these grapevine viruses, which can detect as little as 10 pg of viral RNA. Since then, multiplex PCR has become a widely accepted and used method for detecting grapevine viruses. It has become a valuable tool for vineyard management and grape production, providing a faster and more accurate detection of multiple viruses in a single test.

Based on the results of this study, Gambino et al. developed a multiplex RT-PCR assay for the simultaneous detection of four grapevine viruses: GFLV, GLRaV‑1, GVA, and GVB. Gambino et al. (2006b) successfully designed and optimized a multiplex PCR assay for the simultaneous detection of four grapevine viruses.

This article describes the development of a multiplex PCR protocol capable of detecting five economically important grapevine viruses. As previously mentioned, these viruses cause significant damage to grapevine production worldwide, leading to reduced grape yield and quality. The protocol was developed by designing multiple primer sets specific to the target sequences of each of the five viruses and simultaneously amplifying them in a single reaction. The amplified products were analyzed by gel electrophoresis to determine the presence or absence of the target viruses. The researchers optimized the multiplex PCR protocol using different viral isolates and grapevine varieties. They found that the protocol was highly specific, as it only detected the target sequences of the five grapevine viruses and did not cross-react with other viral or plant DNA.

To design multiplex PCR primers, bioinformatics tools and software were used to identify conserved regions within the genomes of the five grapevine viruses. They also scanned a database of known sequences to ensure that the primers were specific to the target viruses and did not cross-react with other viruses or plant DNA. Primers were then synthesized and tested for their ability to amplify target sequences in a multiplex PCR reaction. Researchers have compared the multiplex PCR protocol to traditional virus detection methods such as serological indexing.

They found that multiplex PCR was faster, more sensitive, and more specific than serological and biological diagnostic methods. This suggests that, like in this study, it has become a valuable tool for grapevine management and production. This multiplex PCR protocol has the potential to be used as a routine diagnostic tool for the detection of grapevine viruses. The protocol can be easily adapted to include additional virus targets and can be used to screen grapevine germplasm for virus-free varieties, monitor grapevine breeding materials for virus contamination, and assist in the management of virus-infected grapevines.

Overall, this article highlights the utility of multiplex PCR for the detection of grapevine viruses and emphasizes its potential as a diagnostic tool for grapevine diseases.

One of the benefits of using multiplex PCR in grapevines is the ability to diagnose viral diseases more quickly and accurately. This method, which can detect multiple viruses in a single reaction, not only saves time but also reduces costs while increasing diagnostic accuracy. Multiplex PCR also allows the detection of viral strains that may not be detected by serological tests, providing a more precise and targeted management strategy. Another advantage of multiplex PCR in grapevines is its potential to help prevent the spread of viral diseases. Multiplex PCR also allows the detection of viral strains that may not be detected by serological tests, providing a more precise and targeted management strategy. Vineyard managers can reduce the economic impact of viral diseases on grape production and promote sustainable viticultural practices by preventing their spread.

Turkey occupies an important position in the world of viticulture and its economic importance is considerable. However, it is not always possible to achieve high yields and high quality products. One of the main reasons for this is that many pathogens, including viruses, bacteria, fungi, viroids and phytoplasmas, can infect grapevines. Early and reliable detection of disease by rapid methods is critical for disease control, sanitation, certification, and quarantine efforts. While ELISA tests are reliable and provide very rapid results, virus detection can sometimes be overlooked, especially in early growing woody plants. In recent years, molecular diagnostic methods such as RT-PCR have been used to rapidly and reliably detect viruses in various plant groups.

In conclusion, the use of multiplex PCR for virus detection in grapevines has several advantages for grape production. This method provides a faster and more accurate diagnosis of viral diseases, aids in preventing the spread of these diseases, and offers valuable insights into the prevalence and distribution of viruses in vineyards. Therefore, the routine application of multiplex PCR as a diagnostic tool for grapevines is predicted to improve vineyard management, increase grape production, enhance harvest quality, promote products for export, and support sustainable viticultural practices.