Isolation and identification of Pithomyces sacchari as a leaf spot pathogen of Helianthus annuus from Pakistan

Sunflower (Helianthus annuus L.) is an important annual crop known for its edible oil. Sunflower is susceptible to many fungal diseases including rusts, rotting, mildews, and leaf spots that result in low crop yield. Presently, infected leaves with leaf spot disease symptoms were collected from Jallo Park, Botanical Garden; University of the Punjab, Canal road, and Johar Town, Lahore for pathogen/s isolation and identification. The identification was executed morphologically as well as genetically by nucleotide sequencing of rDNA using Internal spacer region (ITS) and Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) primers. Morphological characters demonstrated a rapidly growing colony on MEA reaching 5.0-5.5 cm in diameter without zonation. The mycelial growth was rough and cottony white from the front and light pink from the reverse side. Conidia were brown, verruculose, and ellipsoidal with three to five transverse septations and one longitudinal septum ranging from 15 to 30 ± 2 µm in the broadest part. Conidiophores were long, branched, septate, 70-80 × 3-4 µm in size. Based on morphological characteristics, the pathogen was identified as Pithomyces sacchari. In genetic characterization BLAST analysis of the rDNA-ITS region of the pathogen exhibited maximum (100%) homology with other P. sacchari GenBank strains. Similarly, 99% homology was found with partial glyceraldehyde 3-phosphate dehydrogenase (GAPDH). To confirm the pathogenicity, Koch's pathogenicity test was performed by inoculating artificial fungal suspension in pots and plate assays. The emergence of similar disease symptoms and re-isolation of the same pathogens verified Koch's pathogenicity postulates. Conclusively this study confirms the identification of this novel pathogen of sunflowers and necessitates the quick development of management tools.