Multilocus Characterization Reveals an ITS-Defined Haplotype Associated with Pathogenic Variation in Magnaporthe oryzae

Rice blast, caused by Magnaporthe oryzae, is a major constraint to rice production worldwide. This study aimed to investigate the morphological, pathogenic, and molecular diversity of M. oryzae isolates collected from rice-growing regions of Punjab, Pakistan. A total of 125 infected samples were collected, from which 25 representative isolates were selected to represent the observed morphological diversity. Morphological characterization grouped the isolates into four distinct categories based on cultural and conidial characteristics. Pathogenicity assays under greenhouse conditions confirmed that all the tested isolates were pathogenic and fulfilled Koch’s postulates, with significant variation in disease severity and area under the disease progress curve (AUDPC). Isolate RBNN-1 produced the highest disease severity, whereas RBNR-3 produced the lowest. Multilocus molecular identification using ITS, β-tubulin, actin, and calmodulin gene regions confirmed all isolates as M. oryzae. Sequence analysis of the 25 representative isolates revealed high conservation across protein-coding genes, whereas a single nucleotide polymorphism (A→G) in the ITS region identified an ITS-defined haplotype present in seven isolates. Phylogenetic analysis placed these isolates in a well-supported subclade that was consistently associated with a distinct morphological group and higher disease severity under greenhouse conditions. These findings identified an ITS-defined haplotype that was consistently associated with a distinct morphological group and differences in disease severity among the analyzed isolates. This preliminary observation provides a foundation for future studies using higher-resolution genomic approaches to validate and further investigate intraspecific diversity in M. oryzae.