Mitochondrial Marker-Based EvaGreen Real-Time PCR for Specific Detection and Quantification of Exserohilum rostratum in Rice Tissues
DOI:
https://doi.org/10.48048/tis.2026.13546Keywords:
Rice, Leaf spot, Exserohilum rostratum, Setosphaeria rostrata, Real-time PCR, qPCR assay, Mitochondrial markersAbstract
Exserohilum rostratum is a globally distributed ascomycetous fungus with a broad host range, including rice, where it causes brown to reddish-brown leaf spots and grain discoloration. The temperature-dependent development of rice leaf spot disease highlights E. rostratum as a promising model pathogen for studying rice-fungus interactions under elevated temperature conditions. However, disease diagnosis remains challenging due to symptom overlap with other rice-associated fungi and the presence of numerous small, scattered lesions that hinder reliable disease assessment. In addition, sensitive and specific molecular tools for accurate quantification of E. rostratum in rice tissues remain limited. To address this gap, and to our knowledge for the first time, we developed a dye-based real-time PCR assay using EvaGreen chemistry for the specific detection and quantification of E. rostratum in rice tissues. Primers were designed from nuclear loci, including the actin (ACT) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene regions, and from mitochondrial loci, including cytochrome c oxidase subunits 1 and 2 (COX1/2), cytochrome c oxidase subunit 3 (COX3), and apocytochrome b (COB). The COX3 and COB assays showed high specificity for E. rostratum, with no amplification of other common rice-associated fungi. The COX3 assay demonstrated superior performance, with higher amplification efficiency (94.65% - 109.08%) and approximately 7.5-fold greater sensitivity (LOD95 = 61.50 pg μL-1) than the COB assay (59.04% - 75.44% and LOD95 = 464.50 pg μL-1). Both assays enabled successful monitoring of fungal colonization in inoculated rice tissues and showed strong positive correlations between symptom development and pathogen DNA levels. These assays provide accurate and reliable tools for pathogen diagnosis, quantitative disease assessment, cultivar screening, and studies of disease dynamics in the rice–E. rostratum pathosystem.
HIGHLIGHTS
- Established EvaGreen-based qPCR assays for accurate detection and quantification of Exserohilum rostratum in rice.
- COX3 and COB mitochondrial markers provided high specificity against rice-associated fungi.
- The COX3 assay showed higher sensitivity and amplification efficiency than COB.
- The developed assays enables quantitative assessment of rice leaf spot progression in planta.
GRAPHICAL ABSTRACT
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