Comprehensive Genome Analysis on the Novel Species Sphingomonas panacis DCY99T Reveals Insights into Iron Tolerance of Ginseng

Abstract

Plant growth-promoting rhizobacteria play vital roles not only in plant growth, but also in reducing biotic/abiotic stress. Sphingomonas panacis DCY99^T is isolated from soil and root of Panax ginseng with rusty root disease, characterized by raised reddish-brown root and this is seriously affects ginseng cultivation. To investigate the relationship between 159 sequenced Sphingomonas strains, pan-genome analysis was carried out, which suggested genomic diversity of the Sphingomonas genus. Comparative analysis of S. panacis DCY99^T with Sphingomonas sp. LK11 revealed plant growth-promoting potential of S. panacis DCY99^T through indole acetic acid production, phosphate solubilizing, and antifungal abilities. Detailed genomic analysis has shown that S. panacis DCY99^T contain various heavy metals resistance genes in its genome and the plasmid. Functional analysis with Sphingomonas paucimobilis EPA505 predicted that S. panacis DCY99^T possess genes for degradation of polyaromatic hydrocarbon and phenolic compounds in rusty-ginseng root. Interestingly, when primed ginseng with S. panacis DCY99^T during high concentration of iron exposure, iron stress of ginseng was suppressed. In order to detect S. panacis DCY99^T in soil, biomarker was designed using spt gene. This study brings new insights into the role of S. panacis DCY99^T as a microbial inoculant to protect ginseng plants against rusty root disease.

Keywords: Biotic stress; Iron stress; Panax ginseng; Plant growth-promoting rhizobacteria; Sphingomonas.

Figure 1

(A) Visualization of the circular genome of S. panacis DCY99^T. The genome is shown with a base pair (bp) ruler on the outer ring. The S. panacis DCY99^T main chromosome is 5,003,808 bp in size; the plasmid is 319,133 bp in size. The chromosome is arranged clockwise. The two outer circles represent S. panacis DCY99^T CDSs on the forward and reverse strands, respectively. The next circle indicates GC% content and next three circles indicate rRNA, tRNA, and sRNA, respectively. The S. panacis DCY99^T plasmid does not contain the three RNAs. (B) Sphingomonas pan-genome statistics. The Sphingomonas pan-genome can be subdivided into three categories: (i) the core-genome (the set of genes shared by all genomes), (ii) the accessory-genome (the set of genes present in some but not all genomes), and (iii) the unique-genome (genes that are unique to a single genome). The function of each gene in a group was classified using COGs. COG categories are as follows. For cellular processes and signaling, D: cell cycle control, cell division, and chromosome partitioning; M: cell wall/membrane/envelope biogenesis, N: cell motility; O: posttranslational modification, chaperones and protein turnover, T: signal transduction mechanisms, U: intracellular trafficking, secretion, and vesicular transport, V: defense mechanisms. For information storage and processing, J: translation, ribosomal structure, and biogenesis, K: transcription, L: replication, recombination, and repair. For metabolism, C: energy production and conversion, G: carbohydrate transport and metabolism, E: amino acid transport and metabolism, F: nucleotide transport and metabolism, H: coenzyme transport and metabolism, I: lipid transport and metabolism, P: inorganic ion transport and metabolism, Q: secondary metabolites biosynthesis, transport, and catabolism, R: general function prediction only, and S: function unknown. (C) KEGG pathway distribution of the 159 Sphingomonas strains.

Figure 2

(A) Identity of trp operon between S. sp. LK11 and S. panacis DCY99^T. (B) Comparison of indole acetic acid production by the strain DCY99^T without L-tryptophan and with L-tryptophan, 22.4 ± 8.37 µg/mL of indole acetic acid is produced from L-tryptophan. (C) Results of GC-TOF-MS for D-gluconic acid from epithelium and roots of healthy and rusty 10-year-old P. ginseng (Hp, Healthy ginseng epithelium; Hs, Healthy ginseng root; Rp, Rusty-ginseng epithelium; Rs, Rusty-ginseng root). (D) In vitro antagonistic test against C. destructans. C. destructans cultured without and with S. panacis DCY99^T.

Figure 3

(A) Alignment of fieF genes from E. coli K-12 MG1655 and S. panacis DCY99^T; Sphingomonas oligophenolica S5.1; Sphingomonas mail NBRC 15500; Sphingomonas pruni NBRC 15498. (B) Number of heavy metals related genes in four stains of Sphingomonas. (C) Proposed model for the czc efflux system in S. panacis DCY99^T as suggested S. sp. LK11. The czc efflux system consist of cell wall “outer” membrane protein (CzcC); “inner” plasma membrane transport protein (CzcA); membrane fusion protein that extends through both membranes (CzcB) [29].

Figure 4

Pot assessment of iron tolerance in P. ginseng and resistance against the fungal infection. Morphological appearance of P. ginseng in response to iron stress and fungal infection. Pot assay was observed by morphological alterations after 7 days of bacterial inoculation, each pot included five seedlings. (1) Control; (2) P. ginseng seedlings inoculated with S. panacis DCY99^T; (3) Control under 500 mM iron stress; (4) P. ginseng seedlings inoculated with S. panacis DCY99^T under 500 mM iron stress; (5) P. ginseng seedlings inoculated with F. solani; (6) P. ginseng seedlings inoculated with F. solani and S. panacis DCY99^T; (7) P. ginseng seedlings inoculated with F. solani under 500 mM iron stress; (8) P. ginseng seedlings inoculated with F. solani and S. panacis DCY99^T under 500 mM iron stress; (9) P. ginseng seedlings inoculated with I. mors-panacis HB11; (10) P. ginseng seedlings inoculated with I. mors-panacis HB11 and S. panacis DCY99^T; (11) P. ginseng seedlings inoculated with I. mors-panacis HB11 under 500 mM iron stress; (12) P. ginseng seedlings inoculated with I. mors-panacis HB11 and S. panacis DCY99^T under 500 mM iron stress. During iron exposure, the aerial parts and roots of ginseng plants were visibly stressed, however, when the seedlings were primed with S. panacis DCY99^T at the time of iron exposure, iron tolerance was exhibited. But, S. panacis DCY99^T did not fully confer antifungal effect to seedlings.

Figure 5

(A) Catechol meta cleavage and 4-hydroxybenzoate degradation pathways by Sphingomonas species. Catechol meta cleavage pathway consist of four genes: (1) Catechol 2,3-dioxygenase; (2) 2-hydroxymuconic semialdehyde hydrolase; (3) 2-keto-4-pentenoate hydratase; (4) 4-hydroxy-2-oxovalerate aldolase. 4-hydroxybenzoate degradation pathways contain seven genes: (1) P-hydroxybenzoate hydroxylase; (2) Protocatechuate 4,5-dioxygenase; (3) 4-carboxy-2-hydroxymuconate-6-semialdehyde dehydrogenase; (4) 2-pyrone-4,6-dicarboxylic acid hydrolase; (5) 4-oxalomesaconate tautomerase; (6) 4-oxalomesaconate hydratase; (7) 4-carboxy-4-hydroxy-2-oxoadipate aldolase. (B) Position of genes that encode catechol meta cleavage and 4-hydroxybenzoate metabolism from S. panacis DCY99^T genome.

Figure 6

(A) LPS-free gram-negative bacterial cell wall structure of Sphingomonas species. (B) Sphingolipid pathway data from the KEGG database. (C) Heat map based on spt DNA sequences of the 159 Sphingomonas strains for gene of serine palmitoyltransferase from strain DCY99^T. The sequences of spt gene are from the KEGG database. (D) Sequence alignment of the spt primer for S. panacis DCY99^T and 10 different Sphingomonas strains. (E) Quantitative real-time PCR (RT-PCR) analysis of the spt primer with 8 Sphingomonas strains. Lanes-1 and 2, S. panacis DCY99^T and Sphingomonas panaciterrae DCY91; Lane-3, Sphingomonas azotifigens; Lane-4, S. mali; Lane-5, Sphingomonas dokdonensis; Lane-6, Sphingomonas aquatilis; Lane-8, negative control with template DNA, and Lane-9, negative control with primer.