Morphological and Molecular Characterization of Alternaria tenuissima Causing Leaf Spot on Sunflower

Abstract

Leaf spot diseases caused by Alternaria species represent a growing concern in sunflower (Helianthus annuus L.) production systems, resulting in premature defoliation, photosynthetic impairment, and substantial yield losses under conducive environmental conditions. Although several Alternaria species have been associated with foliar diseases of sunflower, accurate species-level identification remains challenging due to overlapping morphological characters among closely related taxa within the Alternaria tenuissima species complex. This study reports a comprehensive morphological and molecular characterization of Alternaria tenuissima (Kunze) Wiltshire isolates recovered from sunflower plants exhibiting characteristic leaf spot symptoms across surveyed production fields. Disease symptoms were systematically described, and fungal isolates were obtained through standard tissue isolation protocols and maintained on potato dextrose agar. Morphological characterization encompassed colony growth characteristics, conidiophore architecture, conidial dimensions, septation patterns, and chain-forming behavior under controlled laboratory conditions. Pathogenicity of representative isolates was confirmed through Koch's postulates using detached leaf and whole-plant inoculation assays. Molecular identification was conducted using multi-locus phylogenetic analyses based on the internal transcribed spacer region, the Alt a1 allergen gene, the RPB2 gene, and the OPA10 locus, enabling reliable placement of isolates within the tenuissima species complex. Phylogenetic trees constructed using maximum likelihood and Bayesian inference methods supported the identity of recovered isolates as A. tenuissima with high bootstrap confidence. Cultural, morphometric, and sequence data collectively established unambiguous species delimitation. These findings provide an essential reference for disease surveillance, resistance screening programs, and the development of targeted fungicide management strategies for Alternaria leaf spot in sunflower production.