Characterization of the Bacterial Microbiome in Natural Populations of Barley Stem Gall Midge, Mayetiola hordei, in Morocco

Abstract

Mayetiola hordei (Kieffer), known as barley stem gall midge, is one of the most destructive barley pests in many areas around the world, inflicting significant qualitative and quantitative damage to crop production. In this study, we investigate the presence of reproductive symbionts, the effect of geographical origin on the bacterial microbiome's structure, and the diversity associated with natural populations of M. hordei located in four barley-producing areas in Morocco. Wolbachia infection was discovered in 9% of the natural populations using a precise 16S rDNA PCR assay. High-throughput sequencing of the V3-V4 region of the bacterial 16S rRNA gene indicated that the native environments of samples had a substantial environmental impact on the microbiota taxonomic assortment. Briefly, 5 phyla, 7 classes, and 42 genera were identified across all the samples. To our knowledge, this is the first report on the bacterial composition of M. hordei natural populations. The presence of Wolbachia infection may assist in the diagnosis of ideal natural populations, providing a new insight into the employment of Wolbachia in the control of barley midge populations, in the context of the sterile insect technique or other biological control methods.

Keywords: 16S rRNA gene; Wolbachia; barley stem gall midge; biological control; next-generation sequencing (NGS); symbiosis.

Figure 1

Maximum likelihood phylogenetic tree of the 13 Wolbachia strains found in barley stem gall midges based on 16S rRNA gene sequences (325 bp) (A: adult, L: larva, R: Rabat, FM: Fes-Meknes). Host species and the GenBank accession numbers are indicated for sequences of representatives of the Wolbachia supergroups A−N. Numbers in each node are bootstrap proportions based on 1000 replications (only values higher than 30% are shown).

Figure 2

Nonmetric multidimensional scaling (NMDS) plot of bacterial communities in barley stem gall midge samples collected from Rabat (purple), Fes-Meknes (blue), Abda (red), and Doukkala (green) (p < 0.001).’d’ stands for the grid dissimilarity scale (d = 0.2 implies that the distance between two grid lines represents approximately 20% dissimilarity between the regions).

Figure 3

Relative abundance of natural barley stem gall midge populations’ microbiota at the phylum (a), class level (b), and heat map (c) of bacterial genera and classes identified in barley stem gall midge populations from the regions of Rabat, Fes-Meknes, Abda, and Doukkala.

Figure 4

Maximum likelihood phylogenetic tree based on Wolbachia-related OTUs (16S rRNA gene) (443 bp full-size alignment): Wolbachia-related sequence acquired from positive Barley stem gall midge samples, as are the other sequences representing the known supergroups from A to Q. The names of the host species and GenBank accession numbers are used to identify Wolbachia sequences. Bootstrap proportions based on 1000 replication are shown by the number in each node.

Figure 5

Co-occurrence and mutual exclusion networks at the family level for OTUs that compose the bacterial communities of Mayetiola hordei natural populations from the four regions: Fes-Meknes (a), Rabat (b), Doukkala (c), and Abda (d). The degree of interaction determines the size of each node. Cases of copresence are represented by green edges, while mutual exclusion is represented by red edges.

Figure 6

Venn diagram of the distribution of bacterial OTUs forming the core microbiome in Mayetiola hordei among the four examined regions.