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Outline
To diagnose an unidentified disease affecting tobacco fields at Daozhen County in Zunyi, and to investigate the phyllosphere microecological characteristics associated with the disease, a comprehensive study was conducted. Phyllosphere fungi were isolated and purified using tissue isolation methods, and identified by molecular biology techniques. The pathogenicity of these phyllosphere fungi was assessed based on Koch′s postulates. Amplicon sequencing was employed to analyze the microbial community structure and diversity, while Biolog ECO microplate assays were used to evaluate the metabolic activity of the phyllosphere microorganisms. A total of 24 fungal isolates belonging to 10 genera were obtained from symptomatic tobacco leaf tissues through tissue isolation. Pathogenicity tests demonstrated that fungi of the genus Diaporthe were pathogenic. Phylogenetic analysis based on ITS, HIS and TEF1-α gene sequences combined with morphological identification revealed that the pathogen responsible for the tobacco leaf spot disease was identified as Diaporthe eres. Amplicon sequencing results indicated that the dominant fungal genera in both healthy and diseased tobacco phyllosphere samples were Bullera (22.89% in healthy and 10.33% in diseased samples), Abrothallus (44.01% and 41.21%). There was no significant difference in fungal diversity index between healthy and diseased tobacco leaves; however, the richness index was lower in diseased samples. Functional prediction suggested that the relative abundance of plant pathogenic fungi was higher in diseased tobacco leaf tissues compared to healthy ones. Carbon substrate utilization assays revealed that the metabolic activity of phyllosphere microorganisms from healthy tobacco leaves was stronger than that of those from diseased leaves. Microorganisms from healthy leaves exhibited high metabolic capability toward 28 carbon sources. In contrast, phyllosphere microorganisms from diseased leaves showed relatively higher metabolic activity toward 12 carbon sources. In conclusion, the previously unknown tobacco leaf spot disease was identified and determined to be caused by Diaporthe eres. Additionally, other pathogenic genera such as Alternaria, Abrothallus, Aspergillus, and Colletotrichum were also present in the disease-associated phyllosphere. These findings provide insight into the fungal microecological characteristics of the phyllosphere associated with the what so called unknown tobacco leaf spot disease.
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| 科 Family | 属数 Number of genus | 种数 Number of species | 占总种数比例 Percentage of total species (%) | 属 Genus | 种数 Number of species | 占总种数比例 Percentage of total species (%) |
|---|---|---|---|---|---|---|
| 鹅膏菌科Amanitaceae | 2 | 11 | 5.26 | 鹅膏菌属 Amanita | 10 | 4.78 |
| 小菇科 Mycenaceae | 2 | 12 | 5.74 | 丝盖伞属 Inocybe | 5 | 2.39 |
| 多孔菌科 Polyporaceae | 8 | 14 | 6.70 | 蜡蘑属 Laccaria | 5 | 2.39 |
| 红菇科 Russulaceae | 3 | 23 | 11.00 | 小皮伞属 Marasmius | 6 | 2.87 |
| 小菇属 Mycena | 11 | 5.26 | ||||
| 光柄菇属 Pluteus | 5 | 2.39 | ||||
| 红菇属 Russula | 17 | 8.13 | ||||
| 栓菌属 Trametes | 5 | 2.39 |
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