Integrated biological and chemical strategies for sustainable management of Nigrospora coryli-induced tobacco leaf spot

Tobacco (Nicotiana tabacum) leaf spot disease caused by fungal pathogens poses a major threat to crop yield and quality worldwide. Recently, Nigrospora coryli was identified as a novel causal agent of tobacco leaf spot in China, yet its biology, ecological interactions, and management strategies remained largely unexplored. In this study, we characterized N. coryli through growth physiology, phyllosphere microbiome analysis, microbial interaction assays, and fungicide sensitivity tests. The pathogen exhibited optimal growth on PDA at 28 °C and pH 6.0, with broad pH tolerance from 4 to 11 and a lethal temperature of 57 °C. Soluble starch and peptone were the preferred carbon and nitrogen sources, respectively. High-throughput sequencing revealed disease-associated phyllosphere shifts, including enrichment of Stenotrophomonas and reduced relative abundance of Pseudomonas in symptomatic leaves, as well as detection of Nigrospora and Sarocladium only in symptomatic samples. Functional screening identified multiple antagonistic microbes, with Aspergillus niger MGM reducing lesion size by 87.6% in planta, whereas co-inoculation with Pseudomonas syringae pv. angulata SLB-5-3 was associated with increased lesion size under controlled conditions. Among eight commercial fungicides, fludioxonil was the most potent (EC₅₀ = 0.0057 µg/mL), followed by the prochloraz-manganese chloride complex, difenoconazole, and fenaminstrobin. Synergistic interactions were observed in binary mixtures: fludioxonil + difenoconazole (1:4, SR = 1.7360), fludioxonil + fenaminstrobin (1:1, SR = 1.5365), and difenoconazole + fenaminstrobin (1:2, SR = 2.0219). These findings provide a basis for further evaluating candidate microbial antagonists and fungicide combinations for the management of N. coryli-induced tobacco leaf spot.