Acta agriculturae Slovenica, 121/3, 1–8, Ljubljana 2025 doi:10.14720/aas.2025.121.3.21670 Original research article / izvirni znanstveni članek Prevalence and natural impact of major wheat viruses in Azerbaijan Nargiz SULTANOVA 1, 2, Fariz AMIRLI 1, Tarana AGHAYEVA 1, Aynur ARABZADA 1, Mina RASTGOU 3 Received January 27, 2025; accepted September 09, 2025 Delo je prispelo 27. januar 2025, sprejeto 9. september 2025 1 Azerbaijan State Oil and Industry University, Baku, Azerbaijan 2 corresponding author: nargizsultanova05@icloud.com 3 Urmia University, Department of Plant Protection, Faculty of Agriculture, Urmia, Iran Prevalence and natural impact of major wheat viruses in Azerbaijan Abstract: Cereal viruses such as Wheat streak mosaic vi- rus (WSMV), Barley yellow dwarf viruses (BYDV), and Wheat dwarf virus (WDV) often occur alongside other wheat patho- gens, making it difficult to diagnose and manage these diseases effectively. To better understand the current situation with these important DNA and RNA viruses, we collected 157 wheat samples showing virus-like symptoms during the 2022-2024 growing seasons. The samples were taken from wheat-growing areas of Azerbaijan, including Absheron, Jalilabad, and Sha- makhi, and were analysed using ELISA and RT-PCR. During our field surveys, we observed potential signs of viral infec- tions, such as stunted growth, yellowing or streaking of leaves, and reduced grain yields. After extracting total RNA and DNA from the samples, we used specific primers to amplify regions of the viruses’ genomes. DNA fragments of 404 bp, 178 bp, and 550 bp were successfully amplified in 25, 39, and 44 samples infected with BYDV, WSMV, and WDV, respectively. These re- sults provide new insights into the prevalence of these viruses in wheat fields across Azerbaijan and provide essential infor- mation for improving management strategies to protect wheat productivity. Key words: cereal viruses, survey, detection, ELISA, RT- PCR, WSMV, BYDV, WDV Razširjenost in naravni vplivi pomembnejših virusov pšenice v Azerbajdžanu Izvleček: Virusna obolenja žit, kot so virus mozaika pše- nice (Wheat streak mosaic virus, WSMV), virus rumene prit- likavosti ječmena/žit (Barley yellow dwarf viruses, BYDV) in virus pritlikavosti pšenice (Wheat dwarf virus, WDV), se po- gosto pojavljajo skupaj z drugimi patogeni pšenice, kar otežuje natančno diagnostiko in učinkovito obvladovanje teh bolezni. Za boljše razumevanje trenutnega stanja teh pomembnih DNK in RNK virusov smo med rastnima sezonama 2022–2024 zbrali 157 vzorcev pšenice, ki so kazali simptome, podobne virusnim. Vzorce smo odvzeli iz območij pridelave pšenice v Azerbaj- džanu, vključno z Absheronom, Jalilabadom in Šamakhi, ter jih analizirali z uporabo ELISA in RT-PCR. Med terenskimi pregledi smo opazili morebitne znake virusnih okužb, kot so zavrta rast, rumenenje ali progasto obarvanje listov ter zman- jšani pridelek zrn. Po ekstrakciji celotne RNK in DNK smo iz vzorcev s specifičnimi primernimi sekvencami namnožili dele genomov virusov. DNK fragmenti dolžine 404 bp, 178 bp in 550 bp so bili uspešno amplificirani v 25, 39 in 44 vzorcih, oku- ženih z BYDV, WSMV oziroma WDV. Ti rezultati nudijo nove vpoglede v razširjenost teh virusov na poljih pšenice po celot- nem Azerbajdžanu in zagotavljajo ključne informacije za izbol- jšanje strategij upravljanja in zaščite pridelave pšenice. Ključne besede: virusi žit, pregled, odkrivanje, ELISA, RT-PCR, WSMV, BYDV, WDV Acta agriculturae Slovenica, 121/3 – 20252 N. SULTANOVA et al. 1 INTRODUCTION Cereals are a cornerstone of global food produc- tion, providing essential staples for billions of people worldwide. Wheat, rice, maize, and barley are among the most important of these crops, grown in many parts of the world. Growing cereals is no easy task—it requires careful planning and management, including planting, irrigation, fertilization, and pest control. These practices are vital not only for ensuring food security but also for maintaining economic stability, particularly in regions where cereal farming is a key industry. In Azerbaijan, bread wheat (Triticum aestivum L.) has been a staple crop for centuries, and it remains the most widely grown grain in the country. In 2020, Azerbaijan harvested nearly 1.87 million tonnes of wheat, with production continuing to rise. By 2021, wheat output had reached 2.16 million metric tons, with a yield of 32,101 kilograms per hect- are. However, the country’s wheat demand is slightly higher—around 3.2 million tons annually (Figure 1). To meet this growing need, research and technological ad- vancements are helping to sustain wheat yields, ensuring that supply can keep pace with demand. Despite the suc- cesses, wheat farming faces several challenges, with viral diseases being one of the most significant threats. Viral dwarfness (Morca et al., 2024), in particular causes seri- ous damage to wheat crops, leading to reduced yields and lower grain quality. These diseases can result in consid- erable economic losses for farmers, making it essential to find effective ways to manage them. Approaches such as planting resistant varieties and implementing strong biosecurity measures are key to controlling the spread of these viruses. A growing body of research is also fo- cused on the links between crop diseases and climate change. As global temperatures rise and weather patterns shift, the spread and severity of viral plant diseases are also changing. Factors like higher CO2 level, changes in ozone, and more frequent droughts can alter the relation- ship between plants and their pathogens, making some diseases more widespread and more damaging (Mish- chenko et al., 2013; 2014). In Europe alone, more than 30 different viruses are known to affect cereals. These in- clude Wheat streak mosaic virus (WSMV), Barley yellow dwarf viruses (BYDV), and Wheat dwarf virus (WDV). WDV, transmitted by the leafhopper Psammotettix ali- enus (Dahlbom, 1850), poses a major threat to cereals, with the virus spreading faster due to increasing tem- peratures, which extend the activity of the vectors and broaden their range. This results in a higher risk of in- fection across large areas of Europe. In some regions, like Sweden, yield losses can reach between 35-90 % in winter wheat fields, and in southern Finland, losses have been recorded as high as 100 % (Lindblad et al., 2022). As temperatures rise, the infection window is expected to get longer, which could lead to more outbreaks and higher infection rates (Habekub et al., 2009). The grow- ing presence of WDV, alongside Wheat streak mosaic vi- rus (WSMV), is prompting increased research into ways to develop virus-resistant wheat varieties (Pfrieme et al., 2023). WSMV, an RNA virus from the Potyviridae fam- ily, is a major concern for wheat production worldwide. The virus is spread by wheat curl mites (Aceria tosichel- la Keifer, 1969), which pick up the virus while feeding on infected plants and spread it to healthy ones. Simi- larly, BYDVs are also RNA-based viruses transmitted by aphids, such as Rhopalosiphum padi (L., 1758) and Sitobion avenae (Fabricius, 1775), which feed on infected plants and spread the virus to others. These viruses cause symptoms like yellowing and stunting of plants, ultimate- ly reducing yields and impacting a wide range of cereal crops, including wheat, barley, oats, and rye. To reduce the impact of these viruses, understanding their genome structure, transmission methods, and the conditions that drive their spread is crucial. This research is necessary for developing better control strategies. This study aims to assess the prevalence of wheat viruses in Azerbaijan’s pri- mary cereal-growing regions and evaluate the seed trans- mission potential of WSMV isolates. While progress has been made, there is still much to learn about how these viruses spread and how they evolve. Only a few wheat va- rieties with genetic resistance to these diseases have been identified, so there is a strong need for further research. This study examines ongoing efforts to develop reliable diagnostic tools, assess epidemiological risks, and imple- ment effective preventive strategies, which are of critical importance for the timely detection of these viruses, ac- curate risk assessment, and the adoption of appropriate management measures. 2 MATERIALS AND METHODS 2.1 PLANT MATERIAL This study explored the presence and distribution of three major wheat viruses—Wheat streak mosaic vi- rus (WSMV), Barley yellow dwarf viruses (BYDV), and Wheat dwarf virus (WDV)—in Azerbaijan’s main cereal- growing regions: Absheron, Jalilabad, and Shamakhi. Be- tween April and May, and during the early days of June, a total of 157 wheat samples were collected from eight different fields, representing various wheat cultivars. The samples were analysed using a combination of serologi- cal (DAS-ELISA) and molecular (RT-PCR) methods to detect the viruses. The samples were taken from plants Acta agriculturae Slovenica, 121/3 – 2025 3 Prevalence and natural impact of major wheat viruses in Azerbaijan showing clear signs of virus infection, such as mosaic patterns on leaves, rolling, yellowing, stunting, and de- formation. Infected plants also exhibited symptoms like mottling, fewer spikes, and in severe cases, necrosis that could result in the complete death of the plant. Once col- lected, the leaf samples were transported to the laborato- ry and stored at 4 °C until further testing could be carried out. The research was carried out in the Bioadaptation Laboratory at the Institute of Molecular Biology and Bio- technologies, Ministry of Science and Education of the Republic of Azerbaijan. 2.2 DOUBLE-ANTIBODY SANDWICH ENZYME- LINKED IMMUNOSORBENT ASSAY (DAS- ELISA) To identify the possible presence of WSMV, BYDV, and WDV, DAS-ELISA tests were carried out using an- tisera developed by DSMZ (Germany), following the manufacturer’s guidelines. For the ELISA procedure, all buffers were prepared according to the provided instruc- tions. Wheat leaf samples were homogenized at a ratio of 1:5 (w/v) in Tris extraction buffer using a sterile mortar and pestle. Next, 100 µl of the extracts were added to mi- crotiter plate wells pre-coated with 100 µl of antibodies diluted at 1:1000 in carbonate coating buffer. The plates were incubated overnight at 4 °C. After incubation, the wells were rinsed with washing buffer and then treated with 100 µl of alkaline phosphatase-conjugated IgG diluted 1:1000 in conjugate buffer. This step was followed by a 3-hour incubation at 37  °C. The wells were then washed again and incubated with 100 µl of substrate for 1 hour at room temperature. Absorbance was measured at 405 nm using a Stat Fax Microplate Reader (Awareness Technology, USA). Each sample was tested in duplicate, and the results were considered positive if the mean absorbance was at least three times higher than the average reading of negative (healthy) controls. In addition to ELISA, the presence of these viruses was also confirmed using RT-PCR. 2.3 RNA EXTRACTION Total RNA was extracted using a modified CTAB method based on Sambrook et al. (1989). For this, 200 mg of scraped bark tissue from basal nodes, petioles, or midribs, as well as 100 mg of leaf tissue, were processed. Leaf tissue was added to 1 ml of pre-warmed extraction buffer containing β-mercaptoethanol inside sterile ex- traction bags. The samples were incubated at 60  °C for 20 minutes with occasional vortex. An equal volume of chloroform : isoamyl alcohol (24:1) was then added, and the mixture was vortexed for 10 minutes. After centrifu- gation at 10,000 rpm for 15 minutes at 4 °C, the upper aqueous phase was carefully collected. To precipitate RNA, 1/3 volume of lithium chloride (LiCl) was added, and the samples were incubated on ice at 4 °C, followed by centrifugation at 10,000 rpm for 20 minutes at 4 °C. The resulting pellet was resuspended in DEPC-treated water, along with 0.2 volumes of sodium acetate (pH 5.2) and 2 volumes of 100 % ethanol. The mixture was incubated at -20 °C for 2 hours and then centrifuged at 10,000 rpm for 15 minutes. After discarding the supernatant, the pellet was washed with 70 % ethanol, air-dried, and stored at -20  °C or -80  °C for future use. The concentration and purity of the extracted RNA were assessed using a Nano- Drop 2000C Spectrophotometer (Thermo Scientific, Waltham, MA, USA) at 230, 260, and 280 nm, and the A260/A280 and A260/A230 ratios were calculated. Ad- ditionally, RNA integrity was confirmed via 1 % agarose gel electrophoresis, with the RNA bands visualized under UV light after ethidium bromide staining. 2.4 REVERSE TRANSCRIPTION-POLYMERASE CHAIN REACTION (RT-PCR) RT-PCR was performed using virus-specific prim- ers targeting a region of the coat protein (CP) gene (see Table 1 for details). RT-PCR was carried out in two steps using an “Ap- plied Biosystems 2720 Thermal Cycler” (USA). To be- gin, 2 μl of extracted RNA was reverse transcribed into Name Sense sequence [5´-3´] Tm (0C) PCR Fragment size WSMV1 C TGCGGAACTTATCGACAACA 61.4 178 WSMV2 V AATCACACGCTGCCACAATA 56.2 BYDV1 C CCGGCGCTATCTTTATTGAA 62.8 404 BYDV2 V CCATTGGCCTTGTAGAGCAT 57.4 WDV3 C TTTRTCTTTGCTCGTAGCCGAGC 53.3 550 WDV5 V AATAATCGGCATACAAATCAGACC 58.8 Table 1: Primers used for viral DNA amplification. Acta agriculturae Slovenica, 121/3 – 20254 N. SULTANOVA et al. complementary DNA (cDNA) in a final reaction volume of 20 μl. The reaction mix included 2 μl of 10 x RT buffer (containing 0.5 M Tris-HCl, 0.7 M KCl, 0.1 M MgCl2, pH 8), 1 μl of DTT (100 mol μl-1), 1 μl of dNTPs (10 mmol μl-1), 0.5 μl of RNase inhibitor (10 mmol μl-1 ), and 2 μl of reverse primer (100 mmol μl-1). This mixture was incu- bated for one hour at 47 °C, along with 0.5 μl of MMuLV reverse transcriptase (200 U μl-1). For the PCR step, 5 μl of the reverse transcription product was used. The PCR reaction was carried out in a final volume of 20 μl, containing 20 ng of cDNA, 10 mM of each dNTP (Solis BioDyne, Estonia), 1.6 mM MgCl2, 1U of Taq DNA polymerase (Solis BioDyne, Estonia), 0.5 μl of each primer (10 pmol μl-1), and 1X PCR buffer. PCR products were stored at 4 °C or -20 °C and analyzed via electrophoresis on a 1.5 % agarose gel, using 1X Tris- Borate-EDTA (TBE) buffer and staining with ethidium bromide (EB). The DNA bands were visualized using a UV-Gel Doc system (UK), and the size of the PCR prod- ucts was estimated with a 2-log DNA Ladder (NEB, UK). 2.5 TESTING METHOD FOR SEED-TRANSMIT- TED VIRUSES The percentage of virus seed transmission (ST) was calculated using the following formula: ST = (n * 100) / N Where n represents the number of virus-infected plants, confirmed through symptom observation, ELISA, and RT-PCR, and N is the total number of plants grown from virus-infected seeds under controlled, protected soil conditions (Albrechtsen, 2006). Statistical analysis of the experimental data was performed using parametric tests for normal distribution. To ensure more reliable re- sults, advanced statistical software such as R or SPSS was employed for data analysis. These tools provide robust methods for calculating standard deviations, analysing variance, and testing hypotheses, offering greater accu- racy and reproducibility in experimental outcomes. 3 RESULTS AND DISCUSSION Wheat is the second most widely grown cereal grain in the world, after maize, and its global trade ex- ceeds that of any other crop. In 2020, total wheat pro- duction worldwide reached 760 million tons. China, India, and Russia are the top three producers, together accounting for about 41  % of global wheat output. In Azerbaijan, wheat, particularly winter wheat, is vital to the country’s food security. Despite its importance to the national economy, the average wheat yield over the past decade has been around 32,101 kilograms per hect- are (Figure 1). To study the presence of viruses in wheat, 157 samples showing virus-like symptoms such as mild chlorosis, downward leaf rolling, leaf mosaic, streak- ing, distortion, and plant stunting were collected from eight fields in Azerbaijan during the 2022-2024 growing seasons (Figure 2). Yellowing leaves and stunted plants Figure 1: Wheat area, yield, and production in Azerbaijan (Source: State Statistical Committee of the Republic of Azerbaijan, https:// ipad.fas.usda.gov/). Acta agriculturae Slovenica, 121/3 – 2025 5 Prevalence and natural impact of major wheat viruses in Azerbaijan were common signs of viral infections in cereals, with a noticeable reduction in the root system. No significant differences were observed in the occurrence of virus symptoms across regions and fields based on visual as- sessments. Our research, based on serological tests, showed that out of all the samples exhibiting virus-like symp- toms, 24.84 % (39 out of 157) tested positive for WSMV, 28.03 % (44 out of 157) for WDV, and 15.92 % (25 out of 157) for BYDV (Figure 3). Notably, incidence of BYDV increased steadily over the years, with 16.29 % of samples testing positive in 2022, 21.22 % in 2023, and 27.17 % in 2024 (Figure 4). Long-term monitoring of wheat fields from 2022 to 2024, coupled with virus detection through serological and molecular methods, revealed an alternating domi- nance of WSMV, BYDV, and WDV across the study pe- riod. No instances of co-infection with the studied vi- ruses were detected. To further confirm the results from the serological tests, molecular analyses (RT-PCR and PCR) were performed. Phloem scrapings from infected leaves were examined using RT-PCR. Specific prim- ers, BYDV1/BYDV2, WSMV1/WSMV2, and WDV3/ WDV5, were used to amplify a segment of the coat protein gene (CP) for each virus. cDNA was synthe- sized from the total RNA extracted from the samples. The RT-PCR results showed amplification products of 550 bp, 178 bp, and 404 bp, confirming the presence of WDV, WSMV, and BYDV, respectively (Figure 5). Total RNA was extracted from juvenile leaves of 12 different wheat samples. The expected amplification products were 550 bp for WDV, 178 bp for WSMV, and 404 bp for BYDV, confirming the presence of these vi- ruses. Lane M represents the DNA marker (2-Log DNA Ladder, NEB, UK), and lanes 1-13 show the results from Figure 2: Virus-induced symptoms observed on wheat plants during the survey in Jalilabad, including mild chlorosis, down- ward leaf rolling, leaf mosaic, streaking, distortion, and plant stunting. Figure 4: ELISA test results for wheat viruses WSMV, WDV, and BYDV during the 2022-2024 growing seasons. Figure 3: Frequency of wheat virus occurrence in Azerbaijan during the 2022-2024 growing seasons. (A) Map of Azerbaijan with highlighted study regions (Absheron, Jalilabad, Shamakhi), (B) Sample distribution (total n = 157 across 3 regions), (C) Virus detec- tion results (WSMV, WDV, BYDV) with counts and percentages. Acta agriculturae Slovenica, 121/3 – 20256 N. SULTANOVA et al. the tested wheat samples. The RNA and RT-PCR prod- ucts were separated in a 1.5 % agarose gel. A thorough review of global literature on wheat vi- ruses reveals a range of viral pathogens affecting wheat crops, posing significant risks to agricultural productiv- ity and food security. The most extensively studied wheat viruses include Wheat streak mosaic virus (WSMV), Bar- ley yellow dwarf virus (BYDV), and Wheat dwarf virus (WDV), all of which exert widespread impacts on wheat production. BYDVs, in particular, are regarded as the most significant cereal viruses due to their global dis- tribution and their capacity to cause considerable yield losses, estimated at 15–25  % in wheat, barley, and oats (Karaozan & Usta, 2020). Furthermore, these viruses have a remarkably broad host range, infecting not only cereals but also numerous weed species and more than 150 grass varieties occurring in meadows and pastures. Research by Spaar (2008) and Schubert et al. (2015) em- phasizes the economic importance of WSMV, which is transmitted by the wheat curl mite and can lead to sig- nificant yield losses. Studies on BYDV, such as those by Karaozan (2020), highlight its complex transmission mechanisms, involving various aphid species and con- tributing to yellow dwarf disease complexes that affect wheat worldwide (Jones, 2003). Additionally, WDV, first identified in Europe, has been spreading globally, with Lindsten and Vacke (1991) documenting its expansion and its detrimental effects on wheat and barley crops. The spread of WDV has also been linked to the increased movement of infected plant material and environmental changes (Przybył et al., 2020). These studies collectively underscore the need for continuous surveillance, ad- vanced diagnostic techniques, and integrated manage- ment approaches to combat wheat viruses and protect global wheat production (Sahragard et al. 2010; Wosula et al., 2014). Seed-transmitted wheat viruses are of particular concern for wheat production, as they can be passed through infected seeds and establish infections in future generations. WSMV and BYDV are among the key vi- ruses transmitted via seeds, and their impact on wheat yield and quality can result in considerable economic losses (Kashiwabara et al., 2007; Li et al., 2011). Detect- ing and managing these seed-transmitted viruses rely on advanced diagnostic methods, such as serological assays and molecular techniques, which can identify viral path- ogens in seeds (Xie et al., 2017). However, these methods can sometimes overestimate transmission rates, as they may detect inactivated viruses within seed parts. Typi- cally, only viruses capable of infecting the seed embryo can be transmitted to the next generation. However, cer- tain viruses, such as those from the genus Tobamovirus, can survive in or on seeds without entering the embryo and can still infect subsequent plants (Chen et al., 2016). Furthermore, embryo-transmissible viruses can remain in an inactivated state in seed parts like the endosperm or testa, even if the embryo itself is not infected (Elena & Lenske, 2008). Understanding the specific mechanisms and conditions under which these viruses are transmit- ted via seeds is crucial for developing effective control strategies (Baranwal et al., 2019). To investigate seed transmission of WSMV, twelve seeds from WSMV-infected Azamatli cultivar plants were treated with 3  % hydrogen peroxide for 20 min- utes, followed by rinsing with water. The seeds were then sprouted in Petri dishes under moist conditions Figure 5: Agarose gel electrophoresis results showing the RT-PCR detection of WDV (4-6) WSMV (7-8), and BYDV (13) using the primer pairs WDV3/WDV5, WSMV1/WSMV2, and BYDV1/BYDV2 respectively. 1- water; 2,3–negative control, WDV; 9,10– negative control, WSMV; 11,12–negative control, BYDV. М–DNA marker–2-log DNA Ladder (NEB, UK). Acta agriculturae Slovenica, 121/3 – 2025 7 Prevalence and natural impact of major wheat viruses in Azerbaijan and planted into pots with sterile soil in a vector-free greenhouse, with a temperature range of 23-24  °C and illumination of 105–135 µmol m-² s-¹ lux. When the plants reached the 2-4 leaf stage, the number of success- fully grown plants was recorded. The wheat plants were inspected for viral symptoms and tested for WSMV us- ing ELISA. Virus seed transmission (ST) was calculated based on the formula from Albrechtsen (2006). The ELISA results were confirmed by the absence of WSMV symptoms in the wheat plants. It was found that both the grains and leaves of new-generation wheat plants did not contain WSMV antigens, indicating that the virus was not transmitted via seeds. Similar findings were reported in earlier studies (Mishchenko, 2009; Mishchenko et al., 2018; Knudson et al., 2010). Robust seed health testing and certification processes are essential for reducing the spread of seed-transmitted wheat viruses, ensuring the health and productivity of wheat crops (Ellis et al., 2014). The findings of this study highlight critical gaps in exist- ing knowledge, particularly the absence of comparative assessments of agroclimatic factors influencing virus dis- semination across the contrasting regions of Azerbaijan. In addition, the dynamics of co-infections remain under- explored, and there is limited evidence regarding the per- formance of molecular diagnostic tools under local field conditions. By addressing these issues, the present re- search provides an important step toward characterizing the regional determinants of viral disease prevalence in cereal crops under diverse agroecosystems of Azerbaijan. 4 CONCLUSIONS This study highlights the significant impact of vi- ral infections, particularly WSMV, BYDV, and WDV, on wheat crops in Azerbaijan, with a notable increase in the prevalence of BYDV over the three-year study period. Long-term monitoring and the use of advanced diagnos- tic techniques, such as ELISA and RT-PCR, confirmed the presence and dynamics of these viruses, emphasizing the importance of continuous surveillance and diagnos- tic precision for effective management. The absence of seed transmission of WSMV, as demonstrated through controlled experiments, underscores the necessity for robust seed health testing and certification processes to prevent the spread of viral infections through infected seeds. 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