Bacteriophages Roam the Wheat Phyllosphere

Abstract

The phyllosphere microbiome plays an important role in plant fitness. Recently, bacteriophages have been shown to play a role in shaping the bacterial community composition of the phyllosphere. However, no studies on the diversity and abundance of phyllosphere bacteriophage communities have been carried out until now. In this study, we extracted, sequenced, and characterized the dsDNA and ssDNA viral community from a phyllosphere for the first time. We sampled leaves from winter wheat (Triticum aestivum), where we identified a total of 876 virus operational taxonomic units (vOTUs), mostly predicted to be bacteriophages with a lytic lifestyle. Remarkably, 848 of these vOTUs corresponded to new viral species, and we estimated a minimum of 2.0 × 10⁶ viral particles per leaf. These results suggest that the wheat phyllosphere harbors a large and active community of novel bacterial viruses. Phylloviruses have potential applications as biocontrol agents against phytopathogenic bacteria or as microbiome modulators to increase plant growth-promoting bacteria.

Keywords: Triticum aestivum; bacteriophage; phyllosphere; viral metagenomics.

Figure 1

Virus operational taxonomic units (vOTUs) recovered from the four viral metagenomes from winter wheat leaves. Viral metagenomes from flag and penultimate leaves were sequenced without (FL, PL, respectively) and with MDA amplification (FL_mda, PL_mda, respectively). (A) Overview of total assembled contigs, identified viral contigs, and species-rank viral clusters (vOTUs), in terms of the number of contigs, cumulative contig length, and the fraction of mapped reads. (B) Predicted genome completeness of the recovered vOTUs. (C) Venn diagram of the presence of vOTUs in the four viral metagenomic samples.

Figure 2

Microbial taxonomic profile and CRISPR-based vOTU host taxonomy assignment. (A) Relative abundance of the dominant bacterial families on the bacterial fraction. Taxonomy was assessed at the read level from the microbial fraction samples (FL_mf and PL_mf). (B) Host prediction performed by matching the vOTUs against CRISPR spacers either from the Dion CRISPR-spacer database or from the assembled microbial contigs. The y-axis represents bacterial families and the x-axis the number of vOTUs predicted to infect such family. Notably, Moraxellaceae and Streptomicetaceae are not highly abundant families in the microbial fraction. However, they were included in the figure, since ≥15 vOTUs were predicted to infect these bacterial families.

Figure 3

Protein sharing network of wheat phyllosphere vOTUs, viral RefSeq genomes, and IMG/VR viral sequences. (A) Overview of the vConTACT2 protein-profile network, depicting the taxonomic context of phylloviruses. In this network, nodes represent viral sequences, and edges connecting two nodes indicate a shared protein profile. RefSeq nodes are colored according to their taxonomy at the order level. Dashed-line circles indicate the 17 discrete groups of interconnected nodes containing only wheat phyllosphere vOTUs. (B) Zoom-in on the biggest group of interconnected nodes containing only wheat phyllosphere. Nodes are colored according to the assigned vConTACT2 viral cluster, which delineates clusters at approximately the genus level.

Figure 4

(A) Description of the ten top-rank abundant phages of each leaf sample (FL and PL). Taxonomic assignments at genus and family levels were made using vConTACT2 and at species level using nucleotide clustering at >95% identity and >85% sequence coverage. The species cluster size represents the number of viral contigs clustered in a vOTU (species-level). On the other hand, genus cluster size refers to the number of viral species (vOTUs) belonging to the same genus cluster assigned by vConTACT2: (C) Complete, (H) High quality, (M) Medium quality, (L) Low-quality, and (U) Unclassified. * Contigs assembled from a subsampled dataset, ** Viral contig matching a CRISPR spacer predicted from the microbial fraction assembly. Green font indicates the two vOTUs that are part of the same genus cluster. (B) Comparative genomics between the wheat phyllosphere APSE phage and the reference genomes of the APSE-1, APSE-2, APSE-3, and APSE-7 subtypes. The amino acid identity between the YD-repeat toxins is indicated in white numbers, and predicted open reading frames are colored by protein groups.