First report late wilt disease caused by Magnaporthiopsis maydis from maize in Republic of Türkiye

Maize, a major crop in Türkiye’s Eastern Mediterranean region, was surveyed for prevalent fungal diseases by collecting symptomatic plants exhibiting growth retardation, stem weakness, leaf yellowing, premature wilting and spike shrinkage during May–June of 2020–2022. A total of 68.9 hectares was surveyed, and 43 lower stem samples were collected from described symptom exhibiting plants across four fields in the Seyhan-Gökçeler district of Adana province. Root collar and stem samples were surface-sterilized [1.5% sodium hypochlorite (NaOCl) for 5 minutes, followed by two 5-minute rinses with sterile distilled water] and tissue pieces of 4 x 4 mm were incubated on PDA at 24 ± 2 °C (12 h light/dark); after three days, white colonies emerged at tissue edges, later forming black-pigmented, sparse, branched, and collapsed mycelium. Fifteen conidia from each of the 24 single-spore isolates were measured, revealing hyaline, unicellular, oblongs spores with a mean size of 11.4 ± 1.7 µm length and 4.75 ± 0.58 µm width (Samra et al., 1963). Genomic DNA from these isolates was extracted and screened using Magnaporthiopsis maydis-specific primers. A single band of approximately 200 bp and 330 bp in length was observed for the primer pairs A200 (A200a: 5’-CCGACGCCTAAAATACAGGA-3’, A200b: 5’-GGGCTTTTTAGGGCCTTTTT-3’) and CMaflp (CMaflp11: 5-TTTCCTGCGGTGCCAA-3, CMaflp12: 5’-TAATGCGGTTAGCCACTC-3’) respectively (Saleh and Leslie, 2004; Drori et al., 2013; Figure 1). The ITS (primers ITS4–ITS5) and RPB1 (primers RPB1F–RPB1R) regions of three randomly selected isolates were sequenced (Klaubauf et al., 2014; O'Donnell et al., 2015) and submitted to NCBI (ITS: OP890270 to OP890272; RPB1: PQ605222 to PQ605224). A BLAST search revealed that the ITS and RPB1 regions were 100% identical (544 bp and 1018 bp, respectively) to M. maydis. The isolates showed 100% sequence identity with Magnaporthiopsis maydis strain CBS 662.82A for both the RPB1 (GenBank: KM485072) and ITS (GenBank: KM484859) gene regions. Multilocus sequence analysis (MLST) was performed by constructing a phylogenetic tree based on concatenated ITS and RPB1 sequences from the three isolates, alongside related species. The isolates clustered with M. maydis, confirming their identity (Figure 2). Pathogenicity was assessed using the susceptible maize genotype P31G98. Seeds were sown in a 1:1:1 peat, perlite, and sand mixture, and two-week-old seedlings were root-dipped in a 1×10⁶ conidia/mL suspension from 10-day-old M. maydis cultures grown on PDA (Biles & Martyn, 1989). Twenty-four isolates were tested in a controlled climate chamber (26 ± 2 °C, 70% RH, 12 h photoperiod) using three pots per isolate, each with four maize seedlings. A negative control (three pots, 12 plants) was inoculated with sterile distilled water. Four weeks post-inoculation, growth retardation and stem/root necrosis were observed. Re-isolations on PDA confirmed Koch’s postulates, with morphological and molecular identification using A200 and CMaflp primers. The experiments were repeated twice. This study aimed to identify major diseases affecting maize in Eastern Mediterranean region of Türkiye and, for the first time, reported the presence of Magnaporthiopsis maydis (syn. Harpophora maydis), the causal agent of late wilt. The results highlight the critical need for integrated management approaches to sustain maize production and mitigate yield losses.