Spatio-Temporal Variation in the Phyllospheric Microbial Biodiversity of Alternaria Alternata-Infected Tobacco Foliage

Abstract

Phyllospheric microbial composition of tobacco (Nicotiana tabacum L.) is contingent upon certain factors, such as the growth stage of the plant, leaf position, and cultivar and its geographical location, which influence, either directly or indirectly, the growth, overall health, and production of the tobacco plant. To better understand the spatiotemporal variation of the community and the divergence of phyllospheric microflora, procured from healthy and diseased tobacco leaves infected by Alternaria alternata, the current study employed microbe culturing, high-throughput technique, and BIOLOG ECO. Microbe culturing resulted in the isolation of 153 culturable fungal isolates belonging to 33 genera and 99 bacterial isolates belonging to 15 genera. High-throughput sequencing revealed that the phyllosphere of tobacco was dominantly colonized by Ascomycota and Proteobacteria, whereas, the most abundant fungal and bacterial genera were Alternaria and Pseudomonas. The relative abundance of Alternaria increased in the upper and middle healthy groups from the first collection time to the third, whereas, the relative abundance of Pseudomonas, Sphingomonas, and Methylobacterium from the same positions increased during gradual leaf aging. Non-metric multi-dimensional scaling (NMDs) showed clustering of fungal communities in healthy samples, while bacterial communities of all diseased and healthy groups were found scattered. FUNGuild analysis, from the first collection stage to the third one in both groups, indicated an increase in the relative abundance of Pathotroph-Saprotroph, Pathotroph-Saprotroph-Symbiotroph, and Pathotroph-Symbiotroph. Inclusive of all samples, as per the PICRUSt analysis, the predominant pathway was metabolism function accounting for 50.03%. The average values of omnilog units (OUs) showed relatively higher utilization rates of carbon sources by the microbial flora of healthy leaves. According to the analysis of genus abundances, leaf growth and leaf position were the important drivers of change in structuring the microbial communities. The current findings revealed the complex ecological dynamics that occur in the phyllospheric microbial communities over the course of a spatiotemporal varying environment with the development of tobacco brown spots, highlighting the importance of community succession.

Keywords: Alternaria alternata; biolog-eco; high-throughput sequencing; microbial community; microbial functional diversity; tobacco brown spot.

Figure 1

The relative abundance of 16 samples at phylum (top 10) and genus (top 30) levels. Different fungal phyla (A) and genus (B); different bacterial phyla (C) and genus (D). Phyla and genus making up <0.01 of total composition were classified as “Others.”

Figure 2

Venn diagram showing the number of operational taxonomic units (OTUs) detected in healthy and diseased tobacco leaves. Fungal Venn diagram in healthy and diseased groups from three positions (A–C). Bacterial Venn diagram in healthy and diseased groups from three positions (D–F).

Figure 3

Maximum likelihood (ML) tree of 100 most abundant fungal (A) and bacterial (B) genera from the tobacco leaves infected with Alternaria alternata obtained by the analysis of ITS rDNA and 16S rRNA pyrosequencing data.

Figure 4

Pie charts showing the relative abundance of top 5 fungal genus of Alternaria, Phoma, Symmetrospora, Cladosporium, Psiloglonium, and “others” in the three analyzed leaf positions and at three collecting time.

Figure 5

Pie charts showing the relative abundance of top 5 bacterial genus of Pseudomonas, sphingomonas, Pantoea, Ralstonia, Kosakonia, and “others” in the three analyzed leaf positions and at three collecting time.

Figure 6

Non-metric multi-dimensional scaling (NMDS) analysis of the fungal (A) and bacterial (B) communities. The spot represents one sample, the distance between two spots represents the degree of deviation, and the same color represents samples in one group. Stress value is <0.2, indicating that NMDS could accurately reflect the difference of microbial communities among samples.

Figure 7

Relative abundance of fungal functional groups (guilds) contingent on OTU annotation table with disturbance frequency level.

Figure 8

Variation of bacterial functional categories based on qualified sequences analyzed by PICRUSt. The KEGG pathway annotation of genes predictions from 16 samples (A). The cluster heatmap at the KEGG level 2 function (B).

Figure 9

Omnilog units (OUs) of carbon sources in BIOLOG ECO-Plate of microbial communities from different samples. The maximum OU values of 16 samples are shown.