Comparative metataxonamic analyses of seeds and leaves of traditional varieties and hybrids of cucumber (Cucumis sativus L.) reveals distinct and core microbiome

Abstract

Profiling the endophytic microbiome of different tissues and varieties of agricultural crops can help to understand i) the tissue specific and varietal specific microbes associated with the plants ii) their potential role in growth, stress tolerance, disease resistance, and yield of the plants. Comparative microbiome profiling across various varieties and hybrids will also be useful to identify the plant's core microbiome. The main objective of the work is to profile and study the microbiome of traditional varieties in comparison with hybrids of cucumber, which would help to understand the microbiome structure in developing consortia to engineer the microbiome of modern hybrids, for useful phenotypes. Metataxonomic sequencing of bacteria and fungi using 16S rRNA gene and ITS regions respectively were carried out in seed and leaf samples of cucumber traditional varieties and modern hybrids. Among bacteria, Prevotella, Bacteroides, Lactobacillus, Dialester, and Fecalibacterium, and among fungal genera, Pichia, Aspergillus, Phaeoisariopsis, Candida, and Malassezia belonged to the core microbiome of cucumber. Modern hybrids were rich in antibiotic producing and toxic pollutant degrading bacteria. Many of the fungi and bacteria observed in the study are well known plant growth promoting microorganisms and play role in offering disease resistance. Some of the bacteria and fungi have beneficial roles in human gut thus revealing the dietary importance of cucumber. The microbes identified in the current study will be useful starting point to develop a consortia to engineer the cucumber microbiome for growth, yield and stress tolerance traits.

Keywords: Bacteria; Cucumber; Endophytes; Fungi; Metataxonomic; Microbiome; Plant growth promotion.

Fig. 1

α-diversity of the endophytic bacteria and fungi in leaves and seeds of traditional varieties and hybrids of cucumber Based on the Shannon and Chao1 index, samples demonstrate variable evenness and richness of bacterial diversity on the X and Y, respectively. The Kruskal-Wallace test, which has P < 0.05, is also used to determine the statistical significance of the grouping based on source (Seeds of traditional varieties = Peach color; Seeds of hybrids = Blue color and Leaves of traditional varieties = Blue color; Leaves of hybrids = Peach color). Fig. 1A and B represent the endophytic bacterial diversity in seeds using Shannon indexing and box plots; Fig. 1C and D represent the Chao1 and box plots. Fig. 1E and F represent the endophytic bacterial diversity in leaves of varieties using Shannon indexing and box plot. Fig. 1G and H represent the Chao1 and box plots. Fig. 1I and J represent the endophytic fungal diversity in seeds of varieties using Shannon indexing and box plots. Fig. 1K and L represent the Chao1 and box plots. Fig. 1M and N represent the endophytic fungal diversity in leaf tissues of varieties using Shannon indexing and box plots. whereas Fig. 1O and P represent the Chao1 and box plots.

Fig. 2

PCoA of the endophytic bacteria and fungi in leaves and seeds of traditional varieties and hybrids of cucumber Fig. 2A: Endophytic bacteria in seeds. Fig. 2B: Endophytic bacteria in leaves. Fig. 2C: Endophytic fungi in seeds. Fig. 2D: Endophytic fungi in leaves. Samples with different diversity were not clustered. Results are presented as 2D ordination plots based on principle coordinate analysis (PCoA). The corresponding statistical significance is assessed using permutational multivariate analysis of variance (PERMANOVA). Samples displayed on the PCoA plots are colored according to the image legends.

Fig. 3

Heat maps of bacterial and fungal genera in leaves and seeds of traditional varieties and hybrids of cucumber Fig. 3A. Heat map of fungal genera. Fig. 3B. Heat map of bacterial genera. Depending on row z-scores, the heat map is colored. Bright red has a strong correlation (r = 3), while bright green has a strong adverse correlation (r = −3). The samples are categorized in the legends by different colors according to how frequently certain genera occur.

Fig. 4

Heat map of the core microbiome of cucumber (fungi and bacteria). Detection threshold used was 10% for predominance and 0.1% for relative abundance. Fig. 4A: core microbiome – bacteria. Fig. 4B: core microbiome – fungi.

Fig. 5

Heatmap of bacterial metabolism in seeds and leaves of traditional varieties and hybrids of cucumber The heat map depicts the number of OTUs responsible for a specific gene function from both seed and leaf samples. Bright red has a strong correlation (r = 3), while bright blue has a strong adverse correlation (r = −3).

Fig. 6

Network analysis in bacteria and fungi in seed and leaf microbiome of traditional varieties and hybrids of cucumber A. Bacteria in seeds B. Fungi in seeds C. Bacteria in leaves D. Fungi in leaves.

Fig. 7

Box plots indicating the top 10 features of correlation in taxa in cucumber seed and leaf microbiome A. Bacteria in seeds B. Fungi in seeds C. Bacteria in leaves D. Fungi in leaves.

Fig. 8

Circos plot analysis of bacterial and fungal diversity in cucumber microbiome based on OTUs The arcs with a particular colour show the relationship between the relative abundance of the microbial community and the samples. The ribbon-shaped area denotes each variable's contribution and shows a strong positive association. A. Circos plot of bacteria B. Circos plot of fungi.