Genetic Variability Among Isolates of Curvularia lunata and Exserohilum rostratum Causing Brown Leaf Spot of Rice from Pakistan.

Curvularia lunata (C. lunata) and Exserohilum rostratum (E. rostratum) are key pathogens causing Brown Leaf Spot (BLS) in rice across Pakistan. The increasing incidence of BLS may be associated with genetic diversity within these fungal populations. This study aimed to identify the pathogens causing BLS and investigated their genetic variability using molecular techniques. Sixteen C. lunata and six E. rostratum isolates were collected from Punjab and identified through morphological characteristics and internal transcribed spacer (ITS) region amplification. ITS-based phylogenetic analysis showed that C. lunata isolates from Pakistan are genetically distinct from those reported globally. Genetic diversity among C. lunata isolates was evaluated using Inter Simple Sequence Repeat (ISSR), Universal Rice Primer (URP), and Random Amplified Polymorphic DNA (RAPD) markers. All markers showed 100% polymorphism, with RAPD producing the highest number of bands (66) and the highest polymorphism information content (PIC = 0.89). Isolate CL-16 exhibited the greatest genetic diversity based on RAPD analysis. Analysis of Molecular Variance (AMOVA) confirmed significant genetic variation within and among C. lunata populations. For E. rostratum, genetic variability was assessed using ISSR, URP, and Simple Sequence Repeat (SSR) markers, all of which also demonstrated complete polymorphism. ISSR primers generated the highest number of bands (105) with a PIC value of 0.73. Cluster analysis using ISSR grouped the isolates into three clusters at a similarity coefficient of 0.68, with isolate EX-6 from Sialkot being the most genetically diverse. However, AMOVA revealed non-significant variation within and among E. rostratum populations. Overall, ISSR primers were most effective for differentiating E. rostratum isolates, while RAPD were more suitable for characterizing C. lunata. The frequent detection of E. rostratum highlights its emergence as a significant BLS pathogen in Pakistan. The ITS and other molecular markers used in this study effectively characterized the isolates and their genetic variability.