Khan Muhammad Faizan Anwar, Riaz Abid, Ahsan Rafia, Ahmad Ishaq, Ullah Saif, Raza Ahmed, Zakria Muhammad
Crop Diseases Research Institute, National Agricultural Research Center, Islamabad, 45500, Pakistan.
Department of Plant Pathology, PMAS Arid Agriculture University, Rawalpindi, Pakistan.
Curr Microbiol. 2025 Aug 5;82(9):436. doi: 10.1007/s00284-025-04425-5.
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.
新月弯孢霉（C. lunata）和喙突埃里格孢（E. rostratum）是巴基斯坦水稻褐叶斑病（BLS）的主要病原菌。褐叶斑病发病率的上升可能与这些真菌群体内的遗传多样性有关。本研究旨在鉴定引起褐叶斑病的病原菌，并使用分子技术研究其遗传变异性。从旁遮普邦收集了16株新月弯孢霉和6株喙突埃里格孢分离株，并通过形态特征和内转录间隔区（ITS）区域扩增进行鉴定。基于ITS的系统发育分析表明，来自巴基斯坦的新月弯孢霉分离株在基因上与全球报道的分离株不同。使用简单序列重复区间（ISSR）、通用水稻引物（URP）和随机扩增多态性DNA（RAPD）标记评估了新月弯孢霉分离株之间的遗传多样性。所有标记均显示出100%的多态性，其中RAPD产生的条带数量最多（66条），多态性信息含量最高（PIC = 0.89）。基于RAPD分析，分离株CL-16表现出最大的遗传多样性。分子方差分析（AMOVA）证实了新月弯孢霉群体内部和群体之间存在显著的遗传变异。对于喙突埃里格孢，使用ISSR、URP和简单序列重复（SSR）标记评估其遗传变异性，所有这些标记也都显示出完全多态性。ISSR引物产生的条带数量最多（105条），PIC值为0.73。使用ISSR进行聚类分析，在相似系数为0.68时将分离株分为三个聚类，来自锡亚尔科特的分离株EX-6遗传多样性最高。然而，AMOVA显示喙突埃里格孢群体内部和群体之间的变异不显著。总体而言，ISSR引物对区分喙突埃里格孢分离株最有效，而RAPD更适合于表征新月弯孢霉。喙突埃里格孢的频繁检测突出了其作为巴基斯坦一种重要的褐叶斑病病原菌的出现。本研究中使用的ITS和其他分子标记有效地表征了分离株及其遗传变异性。
