Full-Length 16S rRNA and ITS Gene Sequencing Revealed Rich Microbial Flora in Roots of Cycas spp. in China

Abstract

Cycads have developed a complex root system categorized either as normal or coralloid roots. Past literatures revealed that a great diversity of key microbes is associated with these roots. This recent study aims to comprehensively determine the diversity and community structure of bacteria and fungi associated with the roots of two Cycas spp. endemic to China, Cycas debaoensis Zhong & Chen and Cycas fairylakea D.Y. Wang using high-throughput amplicon sequencing of the full-length 16S rRNA (V1-V9 hypervariable) and short fragment ITS region. The total DNA from 12 root samples were extracted, amplified, sequenced, and analyzed. Resulting sequences were clustered into 61 bacteria and 2128 fungal OTUs. Analysis of community structure revealed that the coralloid roots were dominated mostly by the nitrogen-fixer Nostocaceae but also contain other non-diazotrophic bacteria. The sequencing of entire 16S rRNA gene identified four different strains of cyanobacteria under the heterocystous genera Nostoc and Desmonostoc. Meanwhile, the top bacterial families in normal roots were Xanthobacteraceae, Burkholderiaceae, and Bacillaceae. Moreover, a diverse fungal community was also found in the roots of cycads and the predominating families were Ophiocordycipitaceae, Nectriaceae, Bionectriaceae, and Trichocomaceae. Our results demonstrated that bacterial diversity in normal roots of C. fairylakea is higher in richness and abundance than C. debaoensis. On the other hand, a slight difference, albeit insignificant, was noted for the diversity of fungi among root types and host species as the number of shared taxa is relatively high (67%). Our results suggested that diverse microbes are present in roots of cycads which potentially interact together to support cycads survival. Our study provided additional knowledge on the microbial diversity and composition in cycads and thus expanding our current knowledge on cycad-microbe association. Our study also considered the possible impact of ex situ conservation on cyanobiont community of cycads.

Keywords: Cycas; coralloid root; diversity; metagenomics; root microbiome.

Figure 1.

Venn diagram displayed the unique and overlapping OTUs of bacteria (B; red color, bold type) and fungi (F; blue color, bold type) between root types and host species. The total number of OTUs were accounted and compared: (a) between normal and coralloid roots regardless of the host species and (b) between Cycas debaoensis and Cycas fairylakea regardless of root types.

Figure 2.

Alpha diversity analysis of microbiomes in the roots of Cycas debaoensis and Cycas fairylakea. The datasets from three individuals for each root type and host species were pooled. Hence, coralloid and normal roots are coded as CD and ND for Cycas debaoensis, and CF and NF for Cycas fairylakea, respectively. The species richness and diversity of bacteria and fungi among these samples were analyzed with three different diversity indices. T-test revealed a statistically significant difference on the diversity of bacteria between root types (P < 0.05) but not with host species. The difference on the diversity of fungi between root types and host species was not significant (P ⩾ 0.05).

Figure 3.

Principal coordinate analysis (PCoA) of bacteria (a) and fungal (b) communities using unweighted UniFrac metrics (β-diversity). In PCoA of bacteria (a), note the slightly distant point of one coralloid root sample of Cycas fairylakea (CF3) and the overlapping points (marked by asterisk *) of the remaining five coralloid root samples (CD1, CD2, CD3, CF1 and CF2). Normal root samples (NF1 and NF2) from replanted adult individuals of Cycas fairylakea were closed to each other while normal root sample (NF3) from host obtained in natural habitat is closer to normal roots of C. debaoensis from botanic garden. (b) A slight variation based on root type and host species was observed in the PCoA of fungal community.

Figure 4.

Relative number of total reads of bacteria (a) and fungi (b) assigned to taxonomic ranks. Samples in this study were coralloid roots (CD1, CD2, CD3, CF1, CF2, CF3) and normal roots (ND1, ND2, ND3, NF1, NF2, NF3) from Cycas debaoensis and Cycas fairylakea.

Figure 5.

Taxonomic composition and relative abundance of microbes detected in the roots of two Cycas spp. using full-length 16S rRNA for bacteria (a, c) and ITS for fungi (b, d). The datasets from three individuals for each root type and host species were averaged and presented per stacked bar. Abbreviations are as in Figure 2. To give a better view of other prevalent bacterial genera present in coralloid roots, members of Nostocaceae were excluded in the dataset as illustrated in C.

Figure 6.

Cyanobacterial composition between coralloid roots of C. debaoensis (CD1, CD2, CD3) and C. fairylakea (CF1, CF2, CF3) using unweighted unifrac. (a) Histogram showed the total read count of the four cyanobacterial OTUs detected in the coralloid roots of the two host species. OTU2 showed the highest read among the two host species. (b) Transplanted samples of Cycas fairylakea (CF1 and CF3) separated in the principal coordinate analysis (PCoA) while the rest of the coralloid root samples from botanic garden and natural habitat (CD1, CD2, CD3, CF2) clustered together.

Figure 7.

Phylogenetic tree of cyanobacteria from the coralloid roots of Cycas debaoensis and Cycas fairylakea inferred by maximum likelihood (ML) and Bayesian inference (BI) based on 16S rRNA genes. The tree was constructed with 69 sequences of free-living and symbiotic strains of cyanobacteria under Order Nostocales downloaded from GenBank, and includes the sequences (OTU2, OTU6, OTU20, OTU4) from the present study. Bootstrap values and posterior probabilities (if available) are given at the nodes, respectively. Representative cyanobacterial strains reported on cycads were included and marked with black star beside the taxa. The sequences of four cyanobacteria analyzed in this study were shown in bold type. OTU4 was further highlighted with double black star as this was only recorded in Cycas fairylakea (CF3) individual originally planted in natural habitat and transferred to botanical garden.