Metagenomic Analysis of Potential Pathogens and Other Microorganisms in Tobacco Leaves

Tobacco leaf spot is a major challenge for tobacco leaf production, and the phyllosphere of tobacco is the main habitat for many pathogens. In this study, tobacco leaves with typical symptoms were sampled, and morphological and molecular biological methods were used to identify pathogens. Illumina high-throughput sequencing and Biolog-ECO were used to investigate the composition and carbon metabolic capacity of the microorganisms in the tobacco leaves. A total of 24 fungal isolates were obtained, including one taxon each of Diaporthe, Paramyrothecium, Botrytis, Phoma, and Mortierella; six each of Fusarium and Epicoccum; four of Trichoderma; and three of Alternaria. Six genera of bacteria were isolated: Bacillus, Pantoea, Enterobacter, Pseudomonas, Prolinoborus, and Atlantibacter. Pathogenicity tests revealed that four isolates of Epicoccum and three isolates of Alternaria were pathogenic, and the leaf spot symptoms induced by coinfection with members from these two groups were similar to those observed in the field. These pathogens were identified as Epicoccum latusicollum and Alternaria alternata through multigene analysis. High-throughput sequencing analysis showed that the dominant fungi in diseased tobacco tissues were Alternaria and Epicoccum, and the dominant bacteria were Pseudomonas, Paenibacillus, and Pantoea. In carbon source utilization tests, where various carbohydrates were the main carbon sources, the utilization capacity of phyllosphere microorganisms in diseased tobacco leaves was lower than that in healthy leaves. The combined application of culture-dependent and independent methods provided comprehensive insights into plant disease diagnosis and tobacco phyllosphere microorganism community composition and metabolic function.