Morphological and Molecular Characterization of Sclerotinia trifoliorum Causing Crown and Stem Rot on Clover

Abstract

Crown and stem rot symptoms characterized by water-soaked lesions, white cottony mycelial growth, progressive tissue collapse, and plant wilting were observed on clover (Trifolium spp.) cultivated under cool and humid environmental conditions. Diseased plant samples collected from affected fields were used for pathogen isolation and characterization through morphological, pathogenicity, and molecular approaches. Fungal isolates consistently produced fast-growing white colonies that later developed black, irregular sclerotia typical of Sclerotinia trifoliorum. Microscopic examination revealed hyaline, septate hyphae and characteristic hyphal structures consistent with the pathogen’s taxonomic description. Considerable variation in sclerotial size, number, and distribution was observed among isolates obtained from different clover-growing regions. Pathogenicity tests conducted on healthy clover plants reproduced typical crown and stem rot symptoms, including tissue softening, crown decay, and plant collapse, whereas control plants remained symptom-free. Re-isolation of the fungus from inoculated tissues fulfilled Koch’s postulates, confirming its pathogenic nature. Molecular characterization was performed using amplification and sequencing of the internal transcribed spacer (ITS) region and additional conserved gene loci relevant for fungal phylogenetic analysis. Sequence comparison revealed high similarity between the obtained isolates and authenticated S. trifoliorum sequences in international databases. Phylogenetic analysis grouped the isolates within the S. trifoliorum clade with strong bootstrap support, confirming species identity and genetic relatedness among regional populations. Environmental conditions characterized by prolonged soil moisture, low to moderate temperatures, and dense canopy cover strongly favored disease development and pathogen persistence. Severe infections resulted in significant reduction in forage biomass and stand longevity, impacting clover productivity in pasture systems. Integrated morphological and molecular approaches provided reliable identification of the pathogen and improved discrimination from closely related Sclerotinia species.