Analysis of seed-associated bacteria and fungi on staple crops using the cultivation and metagenomic approaches

Abstract

One of the key factors affecting seed quality is microbial communities residing on and in the seeds. In this study, microbial populations of seeds of conventionally and organically produced wheat, barley, and maize were analyzed using two different approaches: the cultivation method and metagenomics. For cultivation, three basic media were used: DG18 (for fungi), and nutrient agar or tryptic soy agar supplemented with cycloheximide or nystatin (for bacteria). Metagenomic sequencing was performed using the Illumina MiSeq platform. A total of 452 bacterial isolates comprising 36 genera and 5 phyla and 90 fungal isolates comprising 10 genera and 3 phyla were obtained from the seed surfaces. Among bacteria, representatives from the genera Bacillus, Pantoea, Paenibacillus, and Curtobacterium predominated, and among fungi, Aspergillus predominated. A total of 142 fungal OTUs and 201 bacterial OTUs were obtained from all the samples. Proteobacteria, Firmicutes, Bacteroides, and Actinobacteria comprised most of the bacterial OTUs, and Ascomycota comprised most of the fungal OTUs. Only 3 fungal OTUs (representatives of Curvibasidium, Venturia, and Dermateaceae) were exclusively present only within seeds and not on the seed surfaces. Barley seeds had the highest microbial load and richness, whereas corn had the lowest. Wheat and barley shared a higher number of OTUs than either of them did with corn with higher overlap between conventionally grown cereals than between organically grown cereals. Some OTUs were farming specific. This study demonstrates that the microbiome of cereal seeds is greatly dependent on the species of the host and is less affected by agricultural practices.

Keywords: Barley; Corn; Culture; Microbiota; NGS; Wheat.

Fig. 1

The most abundant bacterial groups detected by 16S-metagenomics on the surfaces of different seeds. The heat plot presents the relative abundances of bacterial OTUs for barley, wheat, and corn grown either organically (ECO) or conventionally (CONV). Letters before the underscores denote the top taxo*-nomical level that could be assigned to the OTU; g, genus; f, family; o, order. Phylogeny is presented as a neighbor-joining tree constructed from Bray-Curtis dissimilarities between OTU representative sequences

Fig. 2

The most abundant fungal groups found on the surfaces of different seeds. The heat plot presents the relative abundances of fungal OTUs for barley, wheat, and corn produced either organically (ECO) or conventionally (CONV). Letters before the underscores denote the highest taxonomical level that could be assigned to the OTU; s, species; g, genus; f, family. Phylogeny is presented as a neighbor-joining tree constructed from Bray-Curtis dissimilarities between OTU representative sequences

Fig. 3

Shared bacterial and fungal species between different seeds. The Venn diagrams show the number and percentage of shared OTUs between barley, corn, and wheat (a) and the overlap of cultivated bacterial and fungal species (b)