First Report of Leaf Spot of Spinacia oleracea Caused by Alternaria burnsii: Aerobiological Implications and Enzymatic Virulence Factor

Spinacia oleracea L. cultivation in South Asia is severely compromised by leaf spot disease caused by fungal plant pathogens, resulting in significant yield and quality losses. In this study, we report the first molecularly confirmed case of an Alternaria burnsii leaf spot on S. oleracea in Pakistan. Symptomatic S. oleracea leaves exhibiting necrotic lesions with concentric rings were collected during a field survey across Bahawalpur district, Punjab, Pakistan in 2024. After isolation, purification and morphological identification it was identified that it belongs to the Alternaria genus. For the confirmation of species, molecular identification was performed; using the ITS and GAPDH primer revealed that the fungal plant pathogen causing leaf spot of S. oleracea is A. burnsii which was also confirmed by phylogenetic analysis. Koch’s postulates were carried out to confirm pathogenicity on detached leaf assays. To assess the virulence of A. burnsii enzymatic analysis was performed. Notably, enzymatic virulence profiling demonstrated a markedly increased production of polygalacturonase (PG: 16.0 ± 0.8 AU), pectin lyase (PNL: 12.0 ± 0.6 AU) and cellulase (CL: 14.0 ± 0.7 AU) relative to controls (all p < 0.001; LSD = 0.16), with PG having the greatest relative increase. This report expands the known host range for A. burnsii and highlights its two-fold threat: as a bioaerosol disseminable by wind and an enzymatic pathogen. These findings highlight the urgent need for integrated disease management strategies for suppressing leaf spot disease in S. oleracea agroecosystems.