, Maize (Zea mays L.) is one of the most important cereals for human and animal consumption and is grown for grain, forage, and silage in the Trakya region. Maize ranks third following wheat and barley, among cereal species, with 8,500,000 tons of production in Türkiye (Turkish Statistical Institute, 2022). Biotic and abiotic stress factors negatively affect maize production every year, with viruses causing the most serious crop losses in epidemic years. To date, Türkiye’s maize fields have been found to harbor six distinct viruses. These include three potyviruses, sugarcane mosaic virus (SCMV), johnsongrass mosaic virus (JGMV), and maize dwarf mosaic virus (MDMV); as well as maize stripe virus tenuivirus (MSpV); the luteovirus barley yellow dwarf virus-PAV (BYDV-PAV); and a nucleorhabdovirus identified as maize mosaic virus (MMV) (Baloğlu et al., 1991; Ertunc, 2023; İlbağı et al., 2006).
Comovirus is a genus in the Secoviridae family, which is in Picornavirales, a large and diverse order (Thompson et al., 2017). Members of most genera in the Picornavirales infect vertebrates and include viruses such as poliovirus, hepatitis A, and human enteroviruses. Members of the Secoviridae infect only plants, but they are placed in the Picornavirales based on (1) similarity of RNA-dependent RNA polymerase (RdRp) sequence, (2) polyprotein gene expression strategy, (3) presence of a small genome-linked protein (viral genome linked protein, VPg) attached to the 5′ end of the genomic RNA and a poly(A) tail at the 3′ end, and (4) icosahedral, T = 3 virion symmetry (Francki et al., 1991; Sanfaon et al., 2009; Thompson et al., 2017; Valverde and Fulton, 1996). The members of the Secoviridae family distinguish themselves from other members of the Picornavirales order by possessing a bipartite genome comprising two distinct RNAs. RNA1 encodes replication proteins, while RNA2 encodes movement and capsid proteins (Thompson et al., 2014). Comoviruses within the Secoviridae are known to infect only dicotyledonous plants and have narrow host ranges, with most members infecting only a few plant species from the Leguminosae family. Some comoviruses, however, are known to infect cucurbits and solanaceous crops (Maina et al., 2017; Valverde and Fulton, 1996). The typical symptoms of comovirus infection in plants include mosaic and mottle patterns. While comoviruses are readily transmitted by mechanical inoculation, certain leaf-feeding beetles in fields can also transmit the virus; other members of the Secoviridae family are transmitted by aphids, nematodes, and through the infection of a small but significant percentage of seeds (Agrios, 2005). The Comovirus genus comprises 19 definitive species characterized by a bipartite, linear, positive-sense RNA genome consisting of two genomic RNAs, each encapsulated separately in an icosahedral (T = 3) virion 25-35 nm in diameter.
High-throughput sequencing (HTS) represents a groundbreaking molecular method used in plant virus diagnostics (Villamor et al., 2019). This advanced technology enables the rapid sequencing of a vast number of nucleotides with considerable redundancy within a relatively short timeframe. Consequently, HTS has a propensity to reveal genomes that may be new virus strains or species. As of this writing, the International Committee on Taxonomy of Viruses lists 19 species of comoviruses, of which Comovirus vignae (cowpea mosaic virus, CPMV), Comovirus trifolii (red clover mottle virus, RCMV), Comovirus siliquae (bean pod mottle virus, BPMV), Comovirus severum (cowpea severe mosaic virus, CPSMV), and Comovirus cucurbitae (squash mosaic virus, SqMV) are among the best-known. The species Comovirus rapae (turnip ringspot virus, TuRSV) infects plants in the Brassicaceae family (Rajakaruna et al., 2007). So far, it has been identified in Chinese cabbage, cauliflower, rocket, and mustard plants, as reported by (Chen et al., 2011; Khandekar et al., 2009; Koloniuk and Petrzik, 2009). TuRSV was initially considered a strain of radish mosaic virus (RaMV) (Rajakaruna et al., 2007) owing to its close sequence relationship (King et al., 2012), but has now been classified as a separate species (Khandekar et al., 2009). This makes sense, as complete nucleotide sequences of RaMV were reported in cruciferous hosts (Komatsu et al., 2007, 2013), while hosts of TuRSV strains have been limited to the Brassicaceae. Isolates of TuRSV have shown no less than 90% sequence identity to each other (Koloniuk and Pertzik, 2009; Komatsu et al., 2008), while RaMV shows only 75.8 and 78.4% amino acid sequence identity for the RdRP and large capsid protein (LCP), to the same regions in TuRSV.
Here, we employed HTS of viral RNAs in maize from sites in Türkiye and obtained the nearly complete and partial genome sequences of a unique isolate of TuRSV from two different maize samples gathered in the Tekirdağ province of Trakya, Türkiye. Our findings lead to the remarkable conclusion that this isolate of TuRSV may be the first known comovirus to infect a monocot.
Materials and Methods
Plant sampling
The plant materials of this study consisted of a total of 28 maize leaf samples, including one or two-leaf samples representing each plant collected from Türkiye and the USA in 2018. Fifteen symptomatic maize leaf samples exhibiting stripe mosaic, mottle, and typically virus-like disease symptoms were collected from the maize fields in Lalapaşa, Pınarhisar, Lüleburgaz, Malkara, and Süleymanpaşa counties of the Tekirdağ, Kırklareli, and Edirne provinces in the Trakya region, Türkiye. Additionally, 13 asymptomatic maize samples, including some infested by vector aphids on the leaves, were collected from maize fields in Ames, Boone, Dekalb, and Newburg counties of the State of Iowa, USA. Furthermore, 145 symptomatic maize leaf samples were collected from the Tekirdağ province of Trakya between 2022 and 2024, and 116 symptomatic canola leaf samples were collected in 2025 to determine virus incidence in Tekirdağ province.
RNA extraction, library preparation, and HTS
For HTS analysis, total RNA was extracted individually from each of a total of 28 maize plants, including 15 symptomatic and 13 asymptomatic leaf samples, using either a DirectZol Miniprep kit (Zymo Research, Irvine, CA, USA) or an RNeasy Plant Mini Kit (Qiagen, Hilden, Germany). The DNA-free total RNA was subjected to ribosomal RNA depletion with the Illumina Ribo-Zero rRNA Removal Kit (Plant Leaf) (Illumina, San Diego, CA, USA) and concentrated using the Zymo RNA Clean & Concentrator-5 kit (Zymo Research). Total RNA was quantified with a Qubit 3.0 fluorometer, and quality assessed with a NanoDrop 2000. Strand-specific cDNA libraries were prepared from ribosome-depleted RNA using the NEBNext Ultra II Directional RNA Library Prep kit (New England Biolabs, Ipswich, MA, USA). The quantity of the cDNA library was also checked 2100 Bioanalyzer System-High Sensitivity DNA Assay (Agilent, Santa Clara, CA, USA). Subsequently, the samples were run on the Illumina HiSeq 3000 (paired-end 150 bp reads) at the Iowa State University DNA Facility, Ames, IA, USA.
Bioinformatic analysis, sequence comparison, and phylogenetic analysis
The raw reads were trimmed with BBMap 38.90 (Bushnell et al., 2017) to remove adapters, barcodes, duplicates, and low-quality sequences (Q > 25). All the samples were assembled using SPAdes v 3.15.1 (Bushmanova et al., 2019). Using trusted contigs from the first assembly, a second assembly was performed with meta viral Spades, contigs were annotated NCBI BLAST 2.11.0+ (Altschul et al., 1990) to the latest ViralDb (last updated February 2021) with a threshold of 50% identity and E < 10−5. Subsequently, the specified contigs were analyzed with NCBI ORF finder, which also uses Smart BLAST and BLASTp. Pairwise alignments of the complete nucleotide and deduced amino acid sequences of TuRSV and related comoviruses were calculated using Bioedit 7.2 (version 7.2.5; https://bioedit.software.informer.com/7.2) (Hall, 1999). Multiple phylogenetic analyses with TuRSV and related comoviruses were performed using 30 and 25 complete and partial nucleotide sequences as well as 23 and 22 complete polyprotein sequences for RNA1 and RNA2, respectively. Tobacco ringspot virus (TRSV) and cherry virus F (CVF) from the Nepovirus and Fabavirus genera in the Secoviridae family were used as an outgroup. The phylogenetic analysis was inferred using a neighbor-joining distance method in Mega 7 by applying the Tamura-Nei model with 1,000 bootstraps (Kumar et al., 1993).
Reverse transcription polymerase chain reaction assay and Sanger sequencing
Reverse transcription polymerase chain reaction (RT-PCR) analysis was performed to determine the presence of TuRSV in maize, canola, and mechanically infected indicator plants, and to confirm HTS data. Total RNA was extracted using a Qiagen plant mini kit (Qiagen). Treatment with DNase I and synthesis of first-strand cDNA with 500 ng RNA and random hexamers were done using a Maxima first-strand cDNA synthesis kit (Thermo Fisher Scientific, Waltham, MA, USA). PCR was carried out with different sets of specific primer pairs (RNA1, 537, 347 bp; RNA2, 692, 433, 416 bp, and control maize phytoene desaturase [PDS] mRNA, 850 bp) designed from assembled contigs of TuRSV-TR59 RNA1 and RNA2 segments (Supplementary Table 1). Each of the 20 μL reaction mixtures included 0.5 μM forward primer, 0.5 μM reverse primer, 10 μL DreamTaq DNA polymerase Mastermix, 2 μL cDNA (100 ng/μL), and 7 μL DEPC-treated water. The thermocycling conditions were as follows: initial denaturation step of 98°C for 10 s; 35 cycles of 98°C for 10 s, 59°C for 30 s (55°C for Maize PDS), 72°C for 3 min; and a final extension step of 72°C for 5 min as well as another cycle condition were performed as follows: initial denaturation step of 95°C for 3 min; 35 cycles of 95°C for 30 s, 54°C for 45 s, 72°C for 1 min; and a final extension step of 72°C for 5 min. After conducting RT-PCR, the amplification products were visualized by gel electrophoresis using 1.5% agarose gels under UV light. Amplicons were purified and extracted using a DNA Gel Extraction Kit (Qiagen). Sanger sequencing was performed at the DNA facility of Iowa State University, USA, and at the DNA facility of Tekirdağ Namık Kemal University, Türkiye.
Reverse transcription quantitative polymerase chain reaction assay
The presence of TuRSV was confirmed by reverse transcription quantitative polymerase chain reaction (RT-qPCR) in two symptomatic maize samples that showed positive reactions in the enzyme-linked immunosorbent assay (ELISA) test. Total RNA was isolated using Qiagen Plant Mini Kit (Qiagen). Direct-Zol Miniprep kit (Zymo Research). A total of 250 mg of each homogenized sample was retained for vortexing with Genie2 at maximum speed for 10 min with ZYMO bashing beads. After removing large components with ZYMO Spin III-F Filter by centrifugation at 8,000 ×g for 1 min, the Direct-Zol Miniprep kit protocol was followed for a complete total RNA isolation. The concentrations of the isolated RNA from the plant materials were determined using the Qubit RNA kit on the Qubit 2.0 fluorometer. cDNA synthesis was performed using a Maxima first-strand cDNA synthesis kit (Thermo Fisher Scientific). Primers for RT-qPCR were designed from the assembled contigs of TuRSV-TR59 RNA1 and RNA2 segments (Supplementary Table 1). The RT-qPCR using SYBR Green qPCR protocol was performed in a PowerAMP Real-Time PCR System (Kogenebiotech, Seoul, Korea) with a total reaction volume of 20 μl. The PCR reaction included 10 μL LightCycler FastStart DNA Master SYBR Green I (Roche, Rotkreuz, Switzerland), 1 μL of each forward and reverse specific primers, 1.5 μL cDNA (100 ng/μL), and 6.5 μL DEPC-treated water. Reaction parameters were set at 95°C for 10 min, 40 cycles of 95°C for 15 s, 60°C for 1 min, and 72°C for 10 s.
Serological detection
A total of 145 symptomatic leaf samples (~ 150 mg/sample) collected in 2022-2024 were tested with polyclonal antibodies (DSMZ, Braunschweig, Germany) for the presence of TuRSV using a double antibody sandwich enzyme-linked immunosorbent assay as described by Clark and Adams (1977).
Mechanical inoculation
A set of indicator test plants, including sweet corn (Zea mays L.), radish (Raphanus sativus L.), and Nicotiana benthamiana L., was used for mechanical transmission of the maize samples infected with TuRSV. Three replications of each indicator test plant, including five plants and a healthy control, were grown in 500 mL pots filled with a sterilized mixture of soil, sand, and compost (1:1:1) and maintained in a growth chamber at 23°C with a 16 h light/8 h dark photoperiod cycle. Symptomatic maize leaf samples found infected with TuRSV were collected from Tekirdağ province of Trakya, Türkiye, in 2022-2024; the infected TuRSV leaves showing mosaic and mottle symptoms were ground 1:10 (w/v) using mortar and pestle in 0.1 M phosphate buffer (pH 7.2) and were used for inoculating indicator plants. The inoculated plants were observed regularly for local or systemic symptom development over a period of 1-3 weeks post-inoculation.
Seed transmission
Maize seeds were harvested from two plants infected with TuRSV marked with rope in the field to investigate transmission by the seed of TuRSV. Five seeds from each plant were grown into 500 mL pots filled with a sterilized mixture of soil, sand, and compost (1:1:1). The plants at the 5-6 leaf stages were harvested to test for the presence of TuRSV by RT-PCR assay.
Results
Symptoms of TuRSV in maize and canola fields
Typical symptoms in maize associated with TuRSV were determined to include stripe mosaic, mottle, and leaf deformations, as shown in Fig. 1. Additionally, it was observed that the infected plants were subjected to systemic damage, smaller cobs and seeds, and drying on the leaves in the end-season period (Fig. 1E). Similarly, mosaic symptoms caused by TuRSV were determined in canola plants (Fig. 1F and G). However, symptomatic plants were mainly observed on the border of the canola fields (Fig. 1H).
Two maize samples from three different locations were identified to contain TuRSV sequences by HTS analysis in 2018, while TuRSV infection was confirmed in two samples from one location by ELISA and PCR assays in 2022-2024. On the other hand, in 2025, eight canola plants gathered from five fields in different locations in the province (Fig. 2) were determined by RT-PCR to have TuRSV infection.
Characterization of the TuRSV-TR59 genome
To identify potential viral pathogenic agents, a total RNA library was constructed from a single leaf from each of the 28 maize plants collected from around the Trakya region (Fig. 1) and the USA, including 13 asymptomatic samples (Iowa, USA) and 15 samples affected by stripe mosaic, mottle, and virus-like disease symptoms (Fig. 2). HTS yielded a total of 372.9 million unique reads. Reads from a sample from Tekirdağ, Trakya (TR59) were assembled into 79,208 contigs (mean: 667.5 nt), with the two contigs having similarity to turnip ringspot virus (TuRSV RNAs 1 and 2), containing a total of 2,078 aligned reads. The BLASTn search of the viral database identified a contig of 6,020 nucleotides with high homology to TuRSV RNA1 (E-value <10-5). The TuRSV RNA1 contig matched 1,860 amino acids at 91.8% identity to a TuRSV cauliflower isolate from Croatia (GenBank accession no. GQ222381). The TuRSV RNA2 contig matched 1,096 amino acids of RNA2 of the cauliflower isolate from Croatia (GenBank accession no. GQ222382) at 88.1% similarity using tBLASTn. We call this isolate TuRSV-TR59. Moreover, another maize sample (TR22) from Tekirdağ, Trakya, contained an 839 nt contig of TuRSV RNA1 sequence (GenBank accession no. OR227625) and a 1,169 nt contig representing TuRSV RNA2 (GenBank accession no. OR227626). We call this isolate TuRSV-TR22. Two reads of TuRSV (TR59 and TR22) contained a small number of reads of radish mosaic virus, MDMV, wheat streak mosaic virus, and red clover necrotic mosaic virus.
The complete genome and partial sequences of the viruses in the subfamily comovirinae of Secoviridae family were retrieved from NCBI for comparative analysis. These translated sequences were aligned against our viral sequence, revealing the highest homology to TuRSV (91.8% tBLASTn), which agrees with earlier BLAST results. Accordingly, the nearly complete genome of TuRSV contains 6,027 nt for RNA1 and 3,920 nt for RNA2, excluding the poly(A) tails (MW720558 for RNA1 and MW720559 for RNA2). RNA1 of TuRSV-TR59 has 298 nt at the 5′ untranslated region (UTR) and 146 nt in the 3′ UTR, while the RNA2 contig has 335 nt at the 5′ UTR and 294 nt at the 3′ UTR. The 5′ UTRs of RNA1 and RNA2 of our viral sequences were 25 nt and 96 nt shorter than other comparable genomes, respectively, while the 3′ UTR of RNA1 and RNA2 were 41 nt and 10 nt shorter (Fig. 3).
RNA1 encodes a single polyprotein (1,860 aa, 211.273 kDa), which includes five functional proteins separated by proteolytic cleavage sites, typical for comoviruses (Chen and Bruening, 1992; Di et al., 1999). The amino acid positions on the polyprotein of the predicted cleavage sites of the TuRSV-TR59 RNA1 and RNA2 are similar to TuRSV (FJ712027) and RaMV as follows: QG, QS, QH in RNA1 and QA, QG in RNA2 sequence as shown in Fig. 4A. We identified a protease cofactor (Pro-Co; 936 nt, 312 aa), helicase (Hel; 1,781 nt, 594 aa), VPg (78 nt, 26 aa), protease peptide (Pro-pe; 630 nt, 210 aa), and RdRp (2,139 nt, 713 aa). RNA-2 contains a single polyprotein (1,096 aa, 123.227 kDa) with three coding regions: the movement protein (MP, 885 nt; 295 aa), the large capsid protein (CP-L; 1,125 nt, 375 aa), and the small capsid protein (CP-S; 251 nt, 251 aa).
While 5′ UTRs are key for controlling protein and RNA synthesis, RNA 1 and RNA 2 were very AU-rich, with GC contents of 35% (RNA 1) and 39% (RNA 2), respectively. Thus, we found few stable RNA secondary structures, though potential structures were found flanking the start codons (Fig. 4B). Guo et al. (2017) found somewhat conserved, AU-rich stem-loops around the start codons in both RNAs of three related comoviruses: BPMV, CPSMV, and CPMV. We also found stem loops flanking the start codons of both TuRSV-TR59 RNAs (Fig. 4B). Each RNA has an AUUAAAU motif in the terminal loop region, which is very similar to sequences in loops of the other comoviral RNAs. TuRSV RNA1 has a ten-base tract identical to that in the terminal loop of CPMV RNA1: GCUUUAUUAA (Fig. 4B). TuRSV RNA1 differs from the other comoviral RNAs in that the terminal loop appears to be very small (Fig. 4B). However, considering that the closing stem is very weak (consisting of four AU pairs), this stem may open in the cell to form a large loop, as in RNA2 and other comoviral RNAs (Guo et al., 2017).
Alignment of the TuRSV 3′ UTRs revealed a common tract of 88 nt with only nine base differences between RNAs 1 and 2 (Fig. 3). Using RNAalifold (Hofacker, 2007) to identify the best secondary structure that both sequences could form, we predict two adjacent bulged stem-loops (Fig. 4C). Crucially, all seven base differences occurring in predicted helices in RNAs 1 and 2 did not affect the base pairing structure, indicating strong support for the conservation of secondary structures. Two other base differences occur in a predicted bulge and do not affect the structure (Fig. 4C). Given the conservation of these structures in both genomic RNAs, they likely play a key role in TuRSV replication.
Sequence comparison and phylogenetic analysis
Sequence comparisons of the TuRSV-TR59 RNA1 sequence revealed that the highest level of nucleotide identity with other published comovirus sequences was 80.56% with DSMZ PV-0355 isolate of TuRSV from Germany. The TuRSV-TR59 RNA2 sequence had the highest nucleotide identity with DSMZ PV-1346 isolate of TuRSV from the Czech Republic (77.77%) (GenBank accession no. ON398518.1). Amino acid multiple sequence alignments of TuRSV-TR59 RNA1 with other comoviruses revealed that the highest level of identity was 92.09% with DSMZ PV-1346 isolate of TuRSV from the Czech Republic. The lowest amino acid identity of TR59 RNA1 between TuRSV isolates was 87.52%, with M12 isolate from Russia. TuRSV-TR59 RNA2 amino acid sequence alignments with other comovirus sequences showed that the highest-level identity was 90.50% with the B isolate of TuRSV from Croatia. The lowest amino acid identity of TR59 RNA2 between TuRSV isolates was 88.13%, with DSMZ-PV-0355 isolate from Germany (GenBank accession no. MW274715.1). Comparing individual polyproteins of the TuRSV-TR59 sequences to other TuRSV/RaMV sequences revealed a 100% identity to the VPg of RaMV sequences. However, the other proteins of the TuRSV-TR59 isolate had different identity values from other published TuRSV sequences, as well as from RaMV sequences.
The nucleotide-based phylogenetic tree clustered TuRSV and RaMV into distinct subclades of the same clade; however, branching differences (Fig. 5A and B) placed TuRSV-TR59 RNA1 isolate beside Czech Republic and Italy isolates, while TuRSV-TR59 RNA2 was more similar to Taiwan and Italy TuRSV isolates.
The complete polyprotein amino acid sequences of TuRSV-TR59 RNA1 and RNA2 were divided into the same clade as other TuRSV polyprotein amino acid sequences. RaMV amino acid sequences clustered closely to TuRSV sequences, but in a distinct subclade. All comoviruses clustered into species-specific clades (Fig. 6A and B), similar to the nucleotide-based phylogenetic result.
Confirmation of HTS sequence by RT-PCR assay
The nucleotide sequences of portions of TuRSV-TR59 RNA1 and RNA2 obtained by HTS in maize samples gathered from Tekirdağ province in 2018 were validated by RT-PCR using primers designed from assembled contigs (Supplementary Table 1) and sequencing the amplicons with Sanger technology. The partial sequences of TuRSV RNA1 (537 bp) and RNA2 (433 bp) were identical to the TR59 maize isolate assembled from HTS. TuRSV RNA1 and RNA2 partial sequences were deposited in GenBank under accession numbers MW767836 and MW767837. To confirm the host plant identity from which the RNA was isolated, primer pairs for a maize PDS gene (Supplementary Table 1) were used to amplify the predicted 850 bp product from the same RNA used for HTS (Supplementary Fig. 1). The PCR product was sequenced and shown to have a 99.438% identity to the maize PDS mRNA (GenBank accession no. MW805425). A partial segment 347 bp long of TuRSV RNA1 had the highest nucleotide identity (82.66%) with TuRSV RNA1 isolate from the Czech Republic. A partial segment 433 bp long of TuRSV RNA2 had the highest nucleotide identity (82.99%) with TuRSV RNA2 isolate from Taiwan.
Serological testing and PCR assay results of surveyed maize samples
ELISA results showed that two of 145 maize leaf samples collected from Tekirdağ province in 2022-2024 were found to be infected with TuRSV, with mean absorbance values (A405) ranging from 1.15 to 0.93 compared to 0.25 in the healthy control. All other maize samples had no positive reaction. To confirm the presence of TuRSV in the two maize samples, RT-qPCR and RT-PCR tests were employed using designed primers from assembled contigs (Supplementary Table 1). The qPCR analysis was assessed by predicting the melting temperature (Tm) of the PCR amplicon as shown in Table 1. Amplification curves are shown in Supplementary Fig. 2.
RT-PCR test results indicated that two symptomatic maize samples and eight canola leaf samples were found to be infected with TuRSV, as shown in Fig. 7.
The amplicons obtained by the RT-PCR assay were Sanger sequenced, and the partial sequences of TuRSV RNA1 and RNA2 were deposited in GenBank under accession numbers PV591078, PV591079, PV624722, and PV624723, respectively. TuRSV RNA1 (537 bp), partial sequences of two samples (416 bp) had 72.76-97.44% and 71.0-95.30% nucleotide sequence identities with Russia and Türkiye isolates. TuRSV RNA2 partial sequences of two maize samples revealed 79.49-97.75% and 78.37-96.62% nucleotide sequence identities with Italy and Türkiye isolates, respectively. The partial sequences of TuRSV RNA1 (GenBank accession no. PV614113) and TuRSV RNA2 (GenBank accession no. PV624721) obtained from the canola isolate showed 72.92-98.93% nucleotide identity with Russia and Türkiye for TuRSV RNA1 and 79.21-97.47% nucleotide identity with Italy, Croatia, and Türkiye isolates for RNA2, respectively.
These results showed that the infection rate was 1.38% in surveyed maize fields in Tekirdağ province. On the other hand, 8 out of 116 canola samples were found to be infected with TuRSV by RT-PCR assay, indicating an infection rate of 6.89% in canola fields. Consequently, the virus incidence was determined to be 8.62% in the surveyed maize and canola fields in Tekirdağ province of Trakya.
Mechanical inoculation results
To determine if TuRSV was infecting maize and not just associated with it, two symptomatic maize plants infected with TuRSV were mechanically inoculated into healthy sweet corn, radish, and N. benthamiana seedlings. Sweet corn and radish indicator plants exhibited mosaic and yellowing symptoms seven days post-inoculation (Fig. 8A and B), and N. benthamiana showed mosaic and mottle symptoms 14 days post-inoculation (Fig. 8C). Symptomatic indicator plants were tested using RT-PCR assay, and positive reactions were obtained for the presence of TuRSV (Fig. 8D).
Moreover, maize plants grown from the seeds of TuRSV-infected plants were tested by RT-PCR. However, the plants obtained from these seeds had no reaction in PCR assay. This result reveals that TuRSV is not readily seed-transmitted.
Discussion
Maize is one of the most widely grown cereals worldwide for human or animal consumption. Many pathogens, abiotic factors, and insects from different families cause considerable yield loss. Viral epidemics cause significant yield and grain quality losses in maize fields. Thus far, six viral pathogens have been identified in maize production areas of Türkiye (Baloğlu et al., 1991; Ertunc, 2023; İlbağı et al., 2006). Many viral diseases cause severe losses in plants such as maize, other cereal species, and grasses from the Poaceae family. However, comoviruses have not been observed to infect monocotyledonous plants such as maize so far. The host range of comoviruses was initially reported to be limited to plants from the Brassicaceae family (Murphy et al., 1995). However, comoviruses have also been shown to infect cucurbits and solanaceous crops (Maina et al., 2017; Valverde and Fulton, 1996). Nineteen virus species have been identified in the comovirus genus so far; however, the number may increase with the recent addition of new viruses using HTS (Villamor et al., 2019). TuRSV was considered as a strain of RaMV until the complete genome sequence was reported by King et al. (2012); however, it has now been classified as a separate species (Khandekar et al., 2009). Comoviruses can be transmitted by mechanical inoculation and through beetles (Smith, 1924; Thompson et al., 2014, 2017), while other members of the Secoviridae family can be transmitted by aphids, nematodes, and through a small but significant percentage of seeds (Agrios, 2005). Rajakaruna et al. (2007) reported that TuRSV can be transmitted mechanically to plants in the Brassicaceae family, exhibiting chlorotic ringspot and line pattern symptoms in turnip. The present study shows that TuRSV can be readily transmitted mechanically to sweet corn, radish, and Nicotiana benthamiana indicator plants, which exhibited characteristic virus symptoms. The symptomatic indicator plants showed positive RT-PCR reactions, confirming the presence of TuRSV, as shown in Fig. 8. The seed transmission experiment, conducted to investigate the possibility of TuRSV transmission by seed in the field, yielded no positive reaction for TuRSV transmission, in line with other studies (Agrios, 2005; Smith, 1924; Thompson et al., 2014, 2017).
To determine the virus incidence, we first investigated the presence of TuRSV in maize fields from 2022 to 2024 and then in canola fields in 2025. TuRSV had infection rates of 1.38% and 6.89% in maize and canola, respectively, in the Tekirdağ province of Trakya. Thus, our results strongly suggest that TuRSV is present and transmissible to maize in the field.
To date, the complete genome sequences of TuRSV have been identified in crucifer species, including cauliflower, turnip, cabbage, Brassica campestris L., Brassica rapa L., mustard, and rocket (Chen et al., 2011; Khandekar et al., 2009; King et al., 2012; Koloniuk and Pertzik, 2009). In addition to these results, our findings reveal that the genome of TuRSV has been identified in maize, which is a novel host of TuRSV in a monocot plant, and the nearly complete sequence diverges significantly from that of other TuRSV isolates. As follows, the nucleotide sequences and amino acid sequences of TuRSV-TR59 RNA1 and RNA2 were 80.56-77.77% and 92.09-90.50%, respectively, compared to other published TuRSV sequences. The amino acid sequences of TuRSV-TR59 RNA1 and RNA2 are much more conserved than the nucleotide sequences. This high conservation of amino acid sequences could be attributed to evolutionary factors such as host shift that influence the virus’ ability to infect a novel host. As reported by Longdon et al. (2014) and Woolhouse et al. (2005), RNA viruses are the most likely group of pathogens to jump from their original host into a novel host species, possibly due to their ability to adapt to new hosts rapidly.
Predicted RNA structures in TuRSV-TR59 resemble those reported recently in other comoviruses. Owing to their location around the polyprotein ORF start codon, the stem-loops that flank the AUG codons might regulate translation. However, as pointed out by Guo et al. (2017) for BPMV, these stem-loops (if the four-base AU helix of RNA1 is disrupted) bear a striking resemblance to the cis-regulatory elements (CRE) of vertebrate viruses in the order Picornavirales, which includes the comoviruses. The CRE is essential for viral RNA replication. It consists of a stem-loop with a large (~13-16 nt) loop containing an AAA tract (Paul and Wimmer, 2015). This tract serves as a template for adding two U’s to the VPg, which then base pair to the poly(A) tail to serve as a primer for RNA synthesis (Paul and Wimmer, 2015). The predicted loops of TuRSV RNAs 1 (with the terminal AU helix disrupted) and 2 are 19 and 13 nt long, respectively, and each has an AAA tract. In various members of the Picornavirales, the CRE can be located in either the coding or noncoding region of the genome; thus, the location flanking the start codons in TuRSV RNAs does not conflict with its functioning as a CRE.
In addition to finding TuRSV RNA associated with maize plants by HTS in 2018, we confirmed its presence in maize and canola fields by mechanical, serological, and molecular tests for virus presence in consecutive years. The obtained partial nucleotide and amino acid sequences of TuRSV of maize and canola isolates were identical to those of TuRSV-TR59 RNA1 and RNA2 sequences. The question arises as to how maize plants acquire TuRSV in the field. Because TuRSV-infected maize fields and canola fields in the Tekirdağ province, specifically in Süleymanpaşa county, are 3 or 4 km apart from each other, as shown in Fig. 2. Additionally, despite the production periods of canola (a winter crop) and maize (a summer crop) differing in the Trakya region, silage maize can be planted early in some fields of Tekirdağ province, coinciding with canola production. Based on the transmission of comoviruses by mechanical inoculation and beetle vectors, it is possible that TuRSV RNA can also be transmitted by leaf-feeding beetles, supporting the notion that TuRSV infects maize. Thus, leaf-feeding beetles may have preferred maize plants for feeding, despite maize being a monocotyledonous plant. As reported by Gergerich (2001) and Hadi et al. (2012), beetles have a large host range to feed on cultivated and wild plants from many families, and they can serve as virus reservoirs. So far, 12 leaf-feeding beetle species from the Chrysomelidae family have been reported in Tekirdağ province and the Trakya region (Bal and Coral, 2022; Özdikmen et al., 2021). In conclusion, TuRSV could be transmitted to maize by leaf-feeding beetles that feed on virus-infected crucifers, brassica plants, or other reservoir hosts in the region. On the other hand, TuRSV may also be transmitted mechanically from virus-infected weeds to maize plants in the field.
To clarify the questions about how maize plants acquire TuRSV, further studies should be conducted to determine the virus etiology and epidemiology in the region. Thus, leaf-feeding beetles and reservoir hosts of TuRSV should also be examined by comprehensive studies in Tekirdağ province and in the Trakya region. In conclusion, our findings suggest that TuRSV may have a broader host range, encompassing both dicotyledonous and monocotyledonous plants, including maize.
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