Complete Genome Sequence of Enterobacter roggenkampii ED5, a Nitrogen Fixing Plant Growth Promoting Endophytic Bacterium With Biocontrol and Stress Tolerance Properties, Isolated From Sugarcane Root

Abstract

Sugarcane is the leading economic crop in China, requires huge quantities of nitrogen in the preliminary plant growth stages. However, the use of an enormous amount of nitrogen fertilizer increases the production price, and have detrimental results on the environment, causes severe soil and water pollution. In this study, a total of 175 endophytic strains were obtained from the sugarcane roots, belonging to five different species, i.e., Saccharum officinarum, Saccharum barberi, Saccharum robustum, Saccharum spontaneum, and Saccharum sinense. Among these, only 23 Enterobacter strains were chosen based on nitrogen fixation, PGP traits, hydrolytic enzymes production, and antifungal activities. Also, all selected strains were showed diverse growth range under different stress conditions, i.e., pH (5-10), temperature (20-45°C), and NaCl (7-12%) and 14 strains confirmed positive nifH, and 12 strains for acdS gene amplification, suggested that these strains could fix nitrogen along with stress tolerance properties. Out of 23 selected strains, Enterobacter roggenkampii ED5 was the most potent strain. Hence, this strain was further selected for comprehensive genome analysis, which includes a genome size of 4,702,851 bp and 56.05% of the average G + C content. Genome annotations estimated 4349 protein-coding with 83 tRNA and 25 rRNA genes. The CDSs number allocated to the KEGG, COG, and GO database were 2839, 4028, and 2949. We recognized a total set of genes that are possibly concerned with ACC deaminase activity, siderophores and plant hormones production, nitrogen and phosphate metabolism, symbiosis, root colonization, biofilm formation, sulfur assimilation and metabolism, along with resistance response toward a range of biotic and abiotic stresses. E. roggenkampii ED5 strain was also a proficient colonizer in sugarcane (variety GT11) and enhanced growth of sugarcane under the greenhouse. To the best of our knowledge, this is the first information on the whole-genome sequence study of endophytic E. roggenkampii ED5 bacterium associated with sugarcane root. And, our findings proposed that identification of predicted genes and metabolic pathways might describe this strain an eco-friendly bioresource to promote sugarcane growth by several mechanisms of actions under multi-stresses.

Keywords: E. roggenkampii; PGPB; endophyte; nitrogen fixation; root colonization; stress; sugarcane; whole-genome sequencing.

FIGURE 1

Intrinsic result of various abiotic stresses of selected endophytic Enterobacter strains growth (A) salt (7–12%), (B) pH (5–10), and (C) temperature (20–45°C).

FIGURE 2

Dendrogram of 16S rRNA gene sequences of selected twenty-three endophytic Enterobacter isolates. The evolutionary distance was calculated by the UPGMA technique. Bootstrap analysis of 1,000 replications is specified as % confidence values for specific branching. Bar indicates % similarity and P. putida, as an outgroup.

FIGURE 3

The dendrogram was created by a nifH gene sequences of the amplified Enterobacter strains by the neighbor-joining method.

FIGURE 4

Scanning electron microscopy (SEM) and CLSM micrographs of most efficient endophytic E. roggenkampii ED5 strain and its colonization in sugarcane plant parts at the root and stem regions. Panels (A,B) is the SEM images showing the morphology of ED5 strain and, (C,D) is the colonization images obtained after the inoculation of ED5 strain in root and stem tissues of sugarcane. Panels (E,F) showing the CLSM micrographs of GFP-tagged endophytic ED5 strain, and (G,H), showing the colonization in the roots and stems of sugarcane by GFP-tagged E. roggenkampii ED5. CLSM images showing the selected strain ED5 in green dots of auto-fluorescence in both root and stem tissues, respectively, and bacterial cells are specified by blue and white arrowheads. Both micrographs confirmed the colonization of inoculated endophytic E. roggenkampii ED5 strain in sugarcane.

FIGURE 5

Circular representation of chromosome and plasmid of endophytic nitrogen-fixing E. roggenkampii ED5 strain isolated from sugarcane root. The inner and innermost rings display the GC content and skew. ORI; the origin of replication in chromosome map. (A,B): A–Z, respectively, show the functional classification of the CDS genes in the chromosome and plasmid with the colors of the COG database and circle 3; different colors show different RNA types.

FIGURE 6

The heat maps of ANI (average nucleotide identity) between strain ED5 and phylogenetically eight closely related species. The ANI value among strain ED5 and E. roggenkampii FDAARGOS was 98.529% and E. roggenkampii ECY546 was 98.507%.

FIGURE 7

Type of secondary metabolites gene clusters in the genome of E. roggenkampii ED5 strain.

FIGURE 8

Types of secretion system present in strain ED5 genome.