Structure vs. chemistry: Alternate mechanisms for controlling leaf microbiomes

Abstract

The analysis of phyllosphere microbiomes traditionally relied on DNA extracted from whole leaves. To investigate the microbial communities on the adaxial (upper) and abaxial (lower) leaf surfaces, swabs were collected from both surfaces of two garden plants, Rhapis excelsa and Cordyline fruticosa. Samples were collected at noon and midnight and at five different locations to investigate if the phyllosphere microbial communities change with time and location. The abaxial surface of Rhapis excelsa and Cordyline fruticosa had fewer bacteria in contrast to its adaxial counterpart. This observation was consistent between noon and midnight and across five different locations. Our co-occurrence network analysis further showed that bacteria were found almost exclusively on the adaxial surface while only a small group of leaf blotch fungi thrived on the abaxial surface. There are higher densities of stomata on the abaxial surface and these openings are vulnerable ports of entry into the plant host. While one might argue about the settling of dust particles and microorganisms on the adaxial surface, we detected differences in reactive chemical activities and microstructures between the adaxial and abaxial surfaces. Our results further suggest that both plant species deploy different defence strategies to deter invading pathogens on the abaxial surface. We hypothesize that chemical and mechanical defence strategies evolved independently for harnessing and controlling phyllosphere microbiomes. Our findings have also advanced our understanding that the abaxial leaf surface is distinct from the adaxial surface and that the reduced microbial diversity is likely a consequence of plant-microbe interactions.

Fig 1. SEM images and bubble charts of microbes, plants and arthropod-related reads on the adaxial and abaxial leaf surfaces.

The bubble sizes correspond to the relative abundance of reads assigned to bacteria, fungi, plant host and arthropods in the comparative metagenomic analyses. Reduction in bacterial read counts observed on the abaxial surface as compared to the adaxial surface in contrast to Fungi, Viridiplantae and Arthropoda reads on the leaf surfaces of (a) R. excelsa and (b) C. fruticosa. The moon and sun symbols refer to the time of sampling (midnight and noon), while the letters refer to the sampling location in S1 Fig. The SEM images were original images captured by Chee Peng Ng and David Liebl from the IMB-IMCB Joint Electron Microscopy Suite (A*STAR Singapore).

Fig 2. Dot plots of species richness using Chao-1 index of the adaxial and abaxial leaf surface.

Approximately 95–98% fewer in bacterial species were observed on the abaxial leaf surface of (a) R. excelsa and (b) C. fruticosa in contrast to their adaxial surface. There was also a decrease in fungal species observed on the abaxial surface of (c) R. excelsa and (d) C. fruticosa. All four dot plots showed statistically significant differences between the adaxial and abaxial surface using Mann Whitney test (S2 Table). The photographs were taken by Balakrishnan N.V. Premkrishnan.

Fig 3. Co-occurrence network analysis of microorganisms on the adaxial and abaxial leaf surfaces.

Of the nine clusters identified, two are highly interconnected (clusters A and B). Microorganisms in both clusters are highly abundant on the adaxial surface but are significantly reduced on the abaxial surface. Cluster A is composed of soil and leaf bacteria while cluster B comprises of wood-rotting fungi, photosynthetic cyanobacteria, thermophilic and radiation-tolerant bacteria. The nodes were selected with a cut-off of Spearman rank correlation coefficient of 0.8 and p-value of 0.01.

Fig 4. Relative abundance of each microbial co-occurrence cluster.

Comparison of the normalised read counts among the nine microbial clusters (A to I) revealed that the orange cluster, composed of leaf blotch fungi, dominates the leaf surfaces of (a) R. excelsa and (b) C. fruticosa. The leaf blotch fungi were twice as abundant on the abaxial surface compared to the adaxial counterpart. The dense clusters A and B network observed in Fig 3 suggest an interaction among the group of leaf, soil, and radio-tolerant bacteria on the adaxial surface. These results further detail the reduction in microbial diversity on the abaxial surface.

Fig 5. Reactive oxygen species assay of adaxial and abaxial leaf surfaces.

A more than 2-fold increase of ROS production was observed on the abaxial surface of (a) R. excelsa. However, this phenomenon was absent in (b) C. fruticosa. The fluorescence intensity readings of the ROS assay are listed in S3 Table. The difference in concentration of ROS between adaxial and abaxial surface of R. excelsa was statistically significant using Welch’s t-test (S5 Table). The photographs were taken by Kenny J.X. Lau.

Fig 6. Surface wettability of leaf surfaces.

The water contact angle was measured from images captured at orthogonal angle. The adaxial and abaxial leaf surfaces of (a) R. excelsa have contact angles of 83° and 95°, respectively. The adaxial and abaxial leaf surfaces of (b) C. fruticosa have contact angles of 102° and 140°, respectively. The abaxial surface is hydrophobic and repels water from its surface. The water droplet images were original images taken by Kenny J.X. Lau using the contact angle meter.