High-Throughput Sequencing-Based Analysis of Rhizosphere and Diazotrophic Bacterial Diversity Among Wild Progenitor and Closely Related Species of Sugarcane (Saccharum spp. Inter-Specific Hybrids)

Abstract

Considering the significant role of genetic background in plant-microbe interactions and that most crop rhizospheric microbial research was focused on cultivars, understanding the diversity of root-associated microbiomes in wild progenitors and closely related crossable species may help to breed better cultivars. This study is aimed to fill a critical knowledge gap on rhizosphere and diazotroph bacterial diversity in the wild progenitors of sugarcane, the essential sugar and the second largest bioenergy crop globally. Using a high-throughput sequencing (HTS) platform, we studied the rhizosphere and diazotroph bacterial community of Saccharum officinarum L. cv. Badila (BRS), Saccharum barberi (S. barberi) Jesw. cv Pansahi (PRS), Saccharum robustum [S. robustum; (RRS), Saccharum spontaneum (S. spontaneum); SRS], and Saccharum sinense (S. sinense) Roxb. cv Uba (URS) by sequencing their 16S rRNA and nifH genes. HTS results revealed that a total of 6,202 bacteria-specific operational taxonomic units (OTUs) were identified, that were distributed as 107 bacterial groups. Out of that, 31 rhizobacterial families are commonly spread in all five species. With respect to nifH gene, S. barberi and S. spontaneum recorded the highest and lowest number of OTUs, respectively. These results were validated by quantitative PCR analysis of both genes. A total of 1,099 OTUs were identified for diazotrophs with a core microbiome of 9 families distributed among all the sugarcane species. The core microbiomes were spread across 20 genera. The increased microbial diversity in the rhizosphere was mainly due to soil physiochemical properties. Most of the genera of rhizobacteria and diazotrophs showed a positive correlation, and few genera negatively correlated with the soil properties. The results showed that sizeable rhizospheric diversity exists across progenitors and close relatives. Still, incidentally, the rhizosphere microbial abundance of progenitors of modern sugarcane was at the lower end of the spectrum, indicating the prospect of Saccharum species introgression breeding may further improve nutrient use and disease and stress tolerance of commercial sugarcane. The considerable variation for rhizosphere microbiome seen in Saccharum species also provides a knowledge base and an experimental system for studying the evolution of rhizobacteria-host plant association during crop domestication.

Keywords: 16S rRNA; diazotroph diversity; nifH; rhizosphere soil; sugarcane.

Figure 1

Schematic diagram of experimental design and analysis that used in the current study. HTS, High-throughput sequencing.

Figure 2

Soil physiochemical properties of rhizospheric soil samples of sugarcane species. Values followed by the same lowercase letters in the same bar are not significantly different according to DMRT 5%. S. officinarum L. cv Badila (BRS), S. barberi Jesw. cv Pansahi (PRS), S. robustum (RRS), S. spontaneum (SRS), and S. sinense Roxb. cv Uba (URS). DMRT, Duncan's Multiple Range test.

Figure 3

Venn diagram showing the OTUs distribution between the sugarcane species soil samples (A) 16S rRNA and (B) nifH gene. S. officinarum L. cv Badila (BRS), S. barberi Jesw. cv Pansahi (PRS), S. robustum (RRS), S. spontaneum (SRS), and S. sinense Roxb. cv Uba (URS). OTUs, operational taxonomic units.

Figure 4

Circular representation of the proportional structure of bacterial communities at the phylum level based on 16s rRNA (A) and nifH gene (B) associated with the sugarcane rhizosphere of different species. Values within the inner circle indicate the number of reads of a phylum within the normalized dataset. S. officinarum L. cv Badila (BRS), S. barberi Jesw. cv Pansahi (PRS), S. robustum (RRS), S. spontaneum (SRS), and S. sinense Roxb. cv Uba (URS).

Figure 5

Heat map showing the abundances of bacterial communities at genus level in the soil samples of sugarcane species (A) 16S rRNA and (B) nifH gene. S. officinarum L. cv Badila (BRS), S. barberi Jesw. cv Pansahi (PRS), S. robustum (RRS), S. spontaneum (SRS), and S. sinense Roxb. cv Uba (URS).

Figure 6

The relative abundances of the top 20 diazotrophs at the genus level in the soil of sugarcane species. S. officinarum L. cv Badila (BRS), S. barberi Jesw. cv Pansahi (PRS), S. robustum (RRS), S. spontaneum (SRS), and S. sinense Roxb. cv Uba (URS). Note: Select species with p < 0.05 to draw a column chart, each column is the relative abundance of different species, where **** means p < 0.0001.

Figure 7

A combination of horizontal multi-sample similarity trees and histograms 16S rRNA (A) and nifH genes (B). On the left is hierarchical clustering between samples based on community composition (Bray-Curtis algorithm), and on the right is a column chart of the sample's community structure. S. officinarum L. cv Badila (BRS), S. barberi Jesw. cv Pansahi (PRS), S. robustum (RRS), S. spontaneum (SRS), and S. sinense Roxb. cv Uba (URS).

Figure 8

Box plot estimated values of gene copies of 16S rRNA (A), and nifH (B) in the soil samples of sugarcane species. Within each box, Small squares denote mean values; boxes extend from the 10th to the 90th percentile of each group's distribution of values. Values followed by the same lowercase letters in the same column are not significantly different according to DMRT 5%. DMRT, Duncan's Multiple Range Test.

Figure 9

Spearman correlation heatmap based on abundance (top 50 genera), diversity indices, gene copy, and soil variables (A) 16S rRNA (B) nifH gene.