RESUMEN
In recent years, STROBY (50% Kresoxim-methyl) has been widely used to control tobacco brown spot in Guizhou Province, China. As a broad-spectrum fungicide, STROBY targets not only phytopathogens, but also affects many other microorganisms including those pathogenic, beneficial, or neutral to the plant hosts. To understand the effects of STROBY on the phyllosphere microbial communities of tobacco leaves during the development of tobacco brown spot, the fungal and bacterial communities of symptomatic and asymptomatic leaves at four time points, before spraying (August 29) and after spraying (September 3, 8, and 13), were investigated using the Illumina high-throughput sequencing. The results showed that STROBY had significant effects on the phyllosphere microbial communities of tobacco leaves. Microbial communities in asymptomatic leaves were more greatly affected than their counterparts in symptomatic leaves, and fungal communities were more sensitive than bacterial communities. Throughout the experiment, the most common genera in symptomatic leaves were Alternaria, Pseudomonas, Pantoea, and Sphingomonas, and in asymptomatic leaves, these were Golubevia and Pantoea. After spraying, the alpha diversity of fungal communities increased in symptomatic leaves and decreased in asymptomatic leaves, while the alpha diversity of bacteria increased in both types of leaves. Beta diversity showed that in asymptomatic leaves, the fungal communities in the first stage was significantly different from the remaining three stages. In contrast, the fungal communities in symptomatic leaves and the bacterial communities in all leaves did not fluctuate significantly during the four stages. Before spraying (August 29), the dominant functions of the fungal community were animal pathogen, endophyte, plant pathogen, and wood saprotroph. Whereas after spraying (September 3, 8, and 13), the proportion of the above fungal functions decreased and the unassigned functions increased, especially in asymptomatic leaves. This study describes the effects of STROBY application and tobacco brown spot presence in shaping the leaf phyllosphere microbial communities, and provides insights into the microbial community effects on tobacco leaves of a strobilurin fungicide.
RESUMEN
In the tobacco phyllosphere, some of the microbes may have detrimental effects on plant health, while many may be neutral or even beneficial. Some cannot be cultivated, so culture-independent methods are needed to explore microbial diversity. In this study, both metagenetic analysis and traditional culture-dependent methods were used on asymptomatic healthy leaves and symptomatic diseased leaves of tobacco plants. In the culture-independent analysis, asymptomatic leaves had higher microbial diversity and richness than symptomatic leaves. Both asymptomatic and symptomatic leaves contained several potentially pathogenic bacterial and fungal genera. The putative bacterial pathogens, such as species of Pseudomonas, Pantoea, or Ralstonia, and putative fungal pathogens, such as species of Phoma, Cladosporium, Alternaria, Fusarium, Corynespora, and Epicoccum, had a higher relative abundance in symptomatic leaves than asymptomatic leaves. FUNGuild analysis indicated that the foliar fungal community also included endophytes, saprotrophs, epiphytes, parasites, and endosymbionts. PICRUSt analysis showed that the dominant functions of the bacterial community in a symptomatic leaf were cellular processes and environmental information processing. In the other five foliar samples, the dominant functions of the bacterial community were genetic information processing, metabolism, and organismal systems. In the traditional culture-dependent method, 47 fungal strains were isolated from 60 symptomatic tobacco leaf fragments bearing leaf spots. Among them, 21 strains of Colletotrichum (29%), Xylariaceae (14%), Corynespora (14%), Pestalotiopsis (10%), Alternaria (10%), Epicoccum (10%), Byssosphaeria (5%), Phoma (5%), and Diaporthe (5%) all fulfilled Koch's postulates and were found to cause disease on detached tobacco leaves in artificial inoculation tests. Symptoms on detached leaves caused by three strains of Corynespora cassiicola in artificial inoculation tests were similar to the original disease symptoms in the tobacco field. This study showed that the combined application of culture-dependent and independent methods could give comprehensive insights into microbial composition that each method alone did not reveal.