The plant material used in this study consisted of maize leaf samples from various varieties exhibiting characteristic symptoms of Maize streak virus (MSV).

The study was conducted in four localities distributed across agro-ecological zones (AEZ) I, V, and VI of Côte d’Ivoire, as indicated in Figure 1 . An agro-ecological zone is a land resource mapping unit based on climate, soil, geology, and/or vegetation cover. Based on these parameters, seven AEZs have been identified in Côte d’Ivoire [11] .

Surveys were conducted in August 2022 and 2023 across three agro-ecological zones (AEZs) in Côte d’Ivoire in particular AEZ I, IV, and VI. The survey covered 11 plots. Upon arrival at the plot, GPS coordinates were recorded using a Garmin Global Positioning System (GPS) device, and the farmers provided information on previous crops and technical itineraries. Sampling was conducted using the “X” method described by Kouakou et al. (2024) [11] . This method involves collecting samples along the two diagonals of the plot. In the field, symptomatic leaf collection was carefully performed using plastic bags to avoid direct contact with the leaves. The collected samples were immediately labeled and transported to the laboratory, where they were finely chopped, preserved in calcium chloride following Bos’s technique, and then stored at 4˚C in a refrigerator [12] .

Total DNA from symptomatic maize leaves was extracted using the cetyltrimethylammonium bromide (CTAB) protocol recommended [13] . Symptomatic leaf samples were ground and homogenized in the presence of DNA extraction buffer. Total DNA was isolated by adding chloroform and precipitating using isopropanol. The detection and quantification of MSV in collected maize leaf samples were performed by amplification and sequencing of the C2 gene in the MSV ORF [14] .

The extracted DNA was quantified using a NanoDrop spectrophotometer (NanoDrop One/OneC). A small volume of distilled water was first used to test the device surface. After calibration, 1 µl of each DNA sample was analyzed.

PCR was performed on the extracted DNA using Go-Taq polymerase (Promega, USA). The reaction mixture consisted of 5 µl of cDNA, 10 mM dNTP (1 µl), 1 µl of each primer pair (sense P1: 5’CCAAAKDTCAGCTCCTCCG3’; antisense P2: 3’TTGGVCCGMVGATGTASAG5’), 0.1 µl of Go-Taq enzyme, and 10 µl of Go-Taq 5x buffer. Sterile water was added to obtain a final volume of 50 µl. The PCR protocol included an initial denaturation cycle at 94˚C for 1 min, followed by 33 denaturation cycles at 94˚C for 1 min, annealing at 50˚C for 1 min, and elongation at 72˚C for 2 min. The final elongation step was conducted at 72˚C for 7 min. The PCR products were stored at 4˚C until electrophoresis.

The amplification products were verified on a 1% (w/v) agarose gel prepared in 0.5x TAE buffer and melted by microwave heating. After cooling, approximately 2 µl of ethidium bromide (EtBr) was added to the agarose solution. The gel was then poured into pre-prepared casting plates. After polymerization, the gel-containing support was placed in an electrophoresis tank filled with 0.5x TAE buffer.

The PCR products (10 µl) were mixed with loading buffer and loaded into gel wells. A 1-kb molecular weight marker was loaded in parallel to estimate the band size. The electrophoresis was conducted at 100 V for 30 min. Amplicons were visualized under UV light using a transilluminator and photographed using a digital camera (Samsung ES74; 14.2 megapixels).

All PCR products of the correct size were sent to Macrogen for sequencing [15] . Bioinformatics analysis involves aligning among sequences. The obtained sequences were edited and aligned using the Molecular Evolutionary Genetics Analysis (MEGA X) software, version 10.05. The obtained sequences were blasted in the NCBI database. Ten sequences from the C2 region of the ORF representing MSV diversity in Africa were downloaded from the NCBI database using accession numbers to study the phylogenetic relationships among isolates.

Nucleotide sequence alignments were performed using the ClustalW alignment algorithm [16] in MEGA X version 10.05. A multiple alignment was generated to produce a distance matrix. From this matrix, a phylogenetic tree was constructed using the appropriate method for the dataset [17] . Progressive sequence alignment was performed according to the branching order.

The evolutionary history was inferred by using the Maximum Likelihood method and Kimura 2-parameter model [18] with 1000 bootstrap repetitions. The tree with the highest log likelihood (−15134.52) is shown. The percentage of trees in which the associated taxa clustered together is shown next to the branches. Initial tree(s) for the heuristic search were obtained automatically by applying Neighbor-Join and BioNJ algorithms to a matrix of pairwise distances estimated using the Maximum Composite Likelihood (MCL) approach, and then selecting the topology with superior log likelihood value. A discrete Gamma distribution was used to model evolutionary rate differences among sites (5 categories (+G, parameter = 0.8188)). The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. This analysis involved 20 nucleotide sequences. There were a total of 2909 positions in the final dataset. Evolutionary analyses were conducted in MEGA X version 10.05 [19] . Only nodes with bootstrap values equal to or greater than 60% were retained.

A total of 20 maize leaf samples, including 15 symptomatic of streak disease and 5 asymptomatic samples, were collected. The main symptoms observed in the plots included light green longitudinal streaks on the leaves and stunted plant growth. Poor grain filling was also noted in infected plants during the fruiting stage ( Figure 2 ).

RT-PCR amplification demonstrated that the symptoms were indeed due to MSV. Figure 3 shows a sample image of the C2 zone of the ORF gene amplification gel from the collected MSV isolates.

The comparison of MSV sequences from Côte d’Ivoire with those from other countries present in the NCBI database showed that the sequences from Côte d’Ivoire are 98% identical to those from Rwanda the phylogenetic tree analysis revealed three monophyletic groups, with Bootstrap values of 100%, 100%, and 95% for groups 1, 2, and 3, respectively ( Figure 4 ). Group 1 consists of MSV 9 Côte d’Ivoire isolates originating from agro-ecological zone I. Group 2 is composed of MSV 4 Côte d’Ivoire isolates from Ferkessédougou (ZAE VI), and Group 3 includes MSV 1 Côte d’Ivoire, MSV 2 Côte d’Ivoire, MSV 3 Côte d’Ivoire, MSV 5 Côte d’Ivoire, MSV 6 Côte d’Ivoire, MSV 7 Côte d’Ivoire, MSV 8 Côte d’Ivoire, and MSV 10 Côte d’Ivoire isolates. MSV 1 Côte d’Ivoire and MSV 2 Côte d’Ivoire isolates originate from Korhogo (ZAE VI), while MSV 3 Côte d’Ivoire, MSV 5 Côte d’Ivoire, and MSV 6 Côte d’Ivoire isolates come from Ferkessédougou (ZAE VI). However, MSV 6 Côte d’Ivoire, MSV 7 Côte d’Ivoire, and MSV 8 Côte d’Ivoire isolates are from Bouaké. Within Group 1, some subgroups were observed. However, these subgroups are not related to the geographical origin of the collection ( Figure 4 ). Moreover, this grouping could be linked to the pathogenic properties of the isolates. It is noteworthy that, in some cases, isolates collected from the same plot are distributed into two different groups. This is the case for MSV 4 Côte d’Ivoire isolates and MSV 3 Côte d’Ivoire, MSV 5 Côte d’Ivoire, and MSV 6 Côte d’Ivoire isolates, which belong to two different monophyletic groups and were collected from the same plot. The tree topology shows a rake-like structure, indicating a lack of phylogenetic structuring. This phylogenetic tree topology of Côte d’Ivoire isolates shows relatively short branches for Group 3 isolates (MSV 1 Côte d’Ivoire, MSV 2 Côte d’Ivoire, MSV 3 Côte d’Ivoire, MSV 5 Côte d’Ivoire, MSV 6 Côte d’Ivoire, MSV 7 Côte d’Ivoire, MSV 8 Côte d’Ivoire, MSV 10 Côte d’Ivoire), whereas the branches of MSV 4 Côte d’Ivoire and MSV 9 Côte d’Ivoire isolates are somewhat longer.