Next Article in Journal
Butyrate Conversion by Sulfate-Reducing and Methanogenic Communities from Anoxic Sediments of Aarhus Bay, Denmark
Next Article in Special Issue
Bacillus telluris sp. nov. Isolated from Greenhouse Soil in Beijing, China
Previous Article in Journal
Expression of Bacillus cereus Virulence-Related Genes in an Ocular Infection-Related Environment
Previous Article in Special Issue
Characterization of a Versatile Plant Growth-Promoting Rhizobacterium Pseudomonas mediterranea Strain S58
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
Microorganisms
Volume 8
Issue 4
10.3390/microorganisms8040608
microorganisms-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles thumb_up
...
Endorse textsms
...
Comment
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

Isolation, Identification and Characterization of Endophytic Bacterium Rhizobium oryzihabitans sp. nov., from Rice Root with Biotechnological Potential in Agriculture

by
Juanjuan Zhao
1,
Xia Zhao
2,
Junru Wang
1,
Qi Gong
1,
Xiaoxia Zhang
1 and
Guishan Zhang
1,*
1
Key Laboratory of Microbial Resources Collection and Preservation, Ministry of Agriculture, Institute of Agricultural Resources and Regional Planning, Chinese Academy of Agricultural Sciences, Beijing 100081, China
2
Beijing Research Institute of Chemical Engineering and Metallurgy, Beijing 101149, China
*
Author to whom correspondence should be addressed.
Microorganisms 2020, 8(4), 608; https://doi.org/10.3390/microorganisms8040608
Submission received: 15 March 2020 / Revised: 6 April 2020 / Accepted: 16 April 2020 / Published: 22 April 2020
(This article belongs to the Special Issue Microbial Isolation and Characterization)
Browse Figures
Versions Notes

Abstract

:
A flagellate, rod–shaped bacterium designated strain M15T was isolated from rice roots. Phylogenetic analysis based on the sequences of the 16S rRNA, housekeeping genes and genomes showed that the isolate belonged to the genus Rhizobium, with the highest 16S rRNA similarity to Rhizobium radiobacter LMG140T (99.64%) and Rhizobium pusense NRCPB10T (99.36%), respectively. The complete genome of the strain M15T has a 59.28% G+C content, and the highest average nucleotide identity (ANI) and DNA-DNA relatedness (DDH) values were obtained with R. radiobacter LMG140T (88.11%, 54.80%), R. pusense NRCPB10T (86.00%, 53.00%) and R. nepotum 39/7T (88.80%, 49.80%), respectively. Plant growth-promoting characteristics tests showed that the strain M15T produced siderophore, 1–aminocyclopropane–1–carboxylate (ACC) deaminase and indole-3-acetic acid (IAA) and also produced some secondary metabolites according to the analysis of the comparative genomes. Based on the data mentioned above, we proposed that the strain M15T represented a novel species of the genus Rhizobium, named Rhizobium oryzihabitans sp. nov. The type strain is M15T (=JCM 32903T  = ACCC 60121T), and the strain M15T can be a novel biofertilizer Rhizobium to reduce the use of synthetic fertilizers for plant growth promotion.

1. Introduction

The genus Rhizobium, belonging to the family Rhizobiaceae within the order Rhizobiales of the class Alphaproteobacteria, was first described by Frank as root and/or stem nodulating bacteria [1]. Generally, most of the Rhizobium species have been isolated from the nodules on leguminous plants with the function of symbiotic nitrogen-fixing. However, some free-living Rhizobium strains have frequently been isolated from soil [2,3,4,5], water [6,7], rhizosphere and plant roots [8]. In addition, several Rhizobium species have been described on the basis of non-symbiotic strains isolated from rice recently, such as Rhizobium oryziradicis, Rhizobium rhizospherae, Rhizobium pseudoryzae, Rhizobium rhizoryzae, and Rhizobium oryzicola [9,10,11,12].
Plant growth-promoting rhizobacteria (PGPR) is a group of rhizosphere bacteria that can enhance plant growth by phosphate solubilization, siderophore production, biological nitrogen fixation, production of 1–aminocyclopropane–1–carboxylate (ACC) deaminase, and production of indole-3-acetic acid (IAA) [13]. Rhizobium is a well–known plant microbiota for nitrogen fixation traits with legume host and non-legume plant growth promoters [14,15,16]. Such as, Rhizobium leguminosarum trifolii E11 is able to promote rice plant growth by phosphate solubilization and IAA production; Bradyrhizobium japonicum can promote the growth of radishes by siderophore production [16], and so on. The mechanisms have been studied thoroughly and used widely in agricultural applications.
In this study, we reported a novel species of Rhizobium isolated from rice roots during the studies of bacterial diversity. The aims of this work were: 1) To isolate and identify the novel Rhizobium strains; 2) To characterize the plant growth-promoting traits of the Rhizobium strains, such as siderophore, ACC deaminase, IAA production, rice plant growth enhancement, and 3) To analyze the genomes and secondary metabolites of the Rhizobium strains against closely related species, and test their possible application as biofertilizers.

2. Materials and Methods

2.1. Strain and Culture Conditions

The endophytic strain M15T was isolated from surface-sterilized rice roots collected from Beijing, China. The isolation and purification were performed on yeast extract mannitol (YMA) medium [17] at 30 ℃. Strains were preserved as glycerol suspension (20%, v/v) at –80 ℃ and at –4 ℃ in freeze-drying ampoules for further characterization.
To study the chemotaxonomic and molecular characteristic, biomass was collected from YMA medium at 30 ℃ for 2 days. The reference strain R. radiobacter LMG140T and R. pusense NRCPB10T were obtained from Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ; Germany). Two reference strains were cultured under the same conditions as strain M15T for comparative analysis.

2.2. Morphological, Physiological and Biochemical Characteristics

Cell morphology was examined using a light microscope (CX21; Olympus). Gram-staining was carried out using the Gram-Stain Kit (solarbio, Beijng, China). Growth features were tested in different temperatures at 4, 15, 25, 30, 37, 40, 45, 50 ℃ and pH 3.0–12.0 (at 1.0 unit intervals) on YMA medium. Salt tolerance was tested on YMA with NaCl concentrations of 0, 1.0, 3.0, 5.0, 7.0, 10.0% (w/v). Motility was observed by semisolid culture–medium (0.4% agar added). Flagellum was observed by transmission electron microscopes (JEM-1400, Japan). Oxidase and catalase activities were determined by using 1% (w/v) tetramethyl–p–phenylenediamine and 3% (v/v) H2O2, respectively. The basic biochemical characteristics were investigated on Biolog GN2 Microplates (Hay-ward, CA, USA) and API–20NE test strips (bioMérieux, Marcy-I’Etoile, France).

2.3. Molecular Studies

Genomic DNA was extracted from pure cultures using Bacteria DNA Kit (Axygen Scientific, Union City, CA) according to the manufacture’s protocol. The 16S rRNA gene was amplified using the universal primers 27F and 1492R according to Lane et al. [18]. The housekeeping genes recA, ropB, atpD were amplified using the methods of Martens et al. [19,20]. The 16S rRNA gene sequences similarity and multiple sequences alignment were analysed using EzTaxon–e Service [21], CLUSTALW [22], respectively. Similarities of housekeeping genes were performed by the National Center for Biotechnology Information Search database BLAST program and multiple sequences alignment was carried out by CLUSTALW. Phylogenetic trees were constructed using MEGA 7.0 software with neighbor–joining methods [23,24,25].
Genome sequencing was carried out using Illumina next–generation sequencing combined with PacBio single–molecule long–read sequencing technology. Software A5–miseq [26], SPAdes [27], HGAP4 [28] and CNAU (V1.6) [29] were used for genome assembly. Prokaryotic Genome Annotation Pipeline (PGAP) was used for genome annotation by NCBI. The genome sequences of strain M15T was submitted to NCBI and the accession number is SAMN14048699. The average nucleotide identity (ANI) values were calculated using ANI Calculator in the EZBioCloud. The DNA–DNA hybridization (DDH) estimates were using GGDC (Genome-to-Genome Distance Calculator) with the BLAST+ (recommended) method [30].

2.4. Fatty Acid Analysis

For cellular fatty acids analysis, strains M15T, R. radiobacter LMG140T and R. pusense NRCPB10T were grown on YMA for 2 days at 30 ℃. Cultures were harvested and fatty acid methyl esters were prepared and separated using methods described by Sasser [31] and were identified with the MIDI Sherlock Microbial Identification system.

2.5. Analysis of Plant Growth-Promoting Characteristics

Production of IAA was analysed according to Glickmann and Dessaux [32]. Quantitative analysis was performed at 50 μg/ml concentrations of tryptophan. A standard graph of IAA in the range of 0–250 μg/ml was used to measure the concentration of IAA produced by strain M15T. Phosphate solubilization was measured on Pikovaskaia’s (PKO) inorganic phosphorus medium at 30 ℃ and observed daily for up to 7 days. The presence of a transparent halo around the colonies demonstrated ability of phosphate solubilization. Siderophore production was detected on chrome azurol S (CAS) agar plates, the color change around the colonies from blue to orange indicated the presence of siderophores [33]. ACC deaminase activity was assayed according to a method described by Perone and Glick [34] with modification. Effects of strain M15T on rice growth was evaluated under liquid culture condition formulated with conventional nutrient composition from the International Rice Research Institute according to Habibi et al. [35] with modification. The high–quality disease–free rice seeds were surface-sterilized. Germinated seeds were growth in pots for 12 days and then transplanted into liquid culture medium inoculated with 5 mL bacterial culture at a cell density of 109 CFU/mL. Uninoculated plants were used as a control. The length and fresh weights of roots and shoots were recorded. All of the experiments were performed in triplicate. The significance of differences between treatments and controls was analyzed using Tukey’s test (P < 0.05).

2.6. Comparative Genomics of Rhizobium Species

Twelve close bacteria belonging to the genus Rhizobium were selected for comparative studies. The 12 bacteria were as follows: Rhizobium nepotum 39/7T, Rhizobium radiobacter LMG140T, Rhizobium pusense NRCPB10T, Rhizobium hainanense CCBAU57015T, Rhizobium freirei PRF81T, Rhizobium miluonense HAMBI2971T, Rhizobium loessense CGMCC1.3401T, Rhizobium mongolense USDA1844T, Rhizobium tibeticum CGMCC1.7071T, Rhizobium grahamii CCGE502T, Rhizobium altiplani BR10423T and Rhizobium oryzihabitans M15T. The Insert Genome Into Species Tree 2.1.10 in KBase was used to constructed a species tree [36]. The phylogenetic tree based on orthologous proteins of the Rhizobium genus was constructed by FastTree [37]. Pan genome analyses were performed by Build Pangenome with OrthoMCL-v2.0 in KBase [36]. A clustering tool MCL was used to cluster protein families. A 50% sequence identity was considered as the cut-off value for the orthologous clustering to obtain the pan and core genome. The virulence factors analysis of strain M15T and R. radiobacter LMG140T, a well known pathogenic species to cause the crown gall disease across thousands of different plant species [38], were performed using Virulence Factors of Pathogenic Bacteria. The secondary metabolite biosynthetic gene clusters of the 12 Rhizobium species were predicted using antiSMASH 5.0 [39]. The synteny maps of the gene clusters were generated using Easyfig [40].

3. Results and Discussion

3.1. Morphological, Physiological and Biochemical Characteristic

Strains M15T was rod and aerobic, flagellate (Supplementary Figure S1), Gram–staining–negative bacteria. Colonies were circular and pearl white on YMA medium at 30 ℃. Growth occurred at 15–50 ℃ (optimal 30 ℃).The pH range for growth was 5.0–12.0 and the tolerance of NaCl was up to 7.0% (w/v), which indicated that strain M15T endowed with inherent capability to cope with high concentration of salt or overly alkaline in soil. Thus, it has the potential for applicaiton in the form of bioinoculants to make the survival of plants easier under extreme saline or alkaline conditions. Strain M15T was observed to be catalase- and oxidase-positive. Other physiological and biochemical characteristics of the novel isolate and reference strains were depicted in Table 1. Strain M15T could be distinguished from R. radiobacter LMG140T in utilization of D-psicose, methyl pyruvate, mono–methyl–succinate, β–hydroxy butyric acid and growth at NaCl (7.0%) and pH11.0. It differed from R. pusense NRCPB10T in assimilation of N–acetyl–D–galactosamine, D–psicose, methyl pyruvate, mono–methy–succinate, formic acid, β–hydroxy butyric acid, α–keto glutaric acid, L–threonine, inosine, uridine, 2,3–butanediol. Discriminative features between the novel isolate and its close relatives are detailed in Table 1.

3.2. Molecular Studies

The genome of strain M15T consists of a circular chromosome of 3,195,345 base pairs, a linearized chromosome of 1,948,381 base pairs and seven plasmids. It was predicted to contain 5670 genes, including 5110 protein-encoding genes, 16 rRNA genes, 59 tRNA genes, 4 ncRNA genes and 483 pseudo genes. The DNA G+C content of strain M15T was 59.28 mol %, which was within the range reported for Rhizobium species (57–66 mol %) [41], implying that it belong to the genus Rhizobium. The ANI between strain M15T and R. nepotum 39/7T, R. radiobacter LMG140T, and R. pusense NRCPB10T were 88.80%, 88.11%, 86.00%, respectively, which is below the referral threshold value (95–96%) for species demarcation suggested by Kim et al. [42] and Richter et al. [43] (Supplementary Table S1).
The DDH between strain M15T and R. radiobacter LMG140T, R. pusense NRCPB10T were 54.80%, 53.00%, respectively, which is lower than the same species threshold of 70% [44,45] (Supplementary Table S2). These results showed that strain M15T represent a novel species of the genus Rhizobium.
The almost complete 16S rRNA gene sequences of strain M15T was determined and subjected to phylogenetic analysis. Similarity search in EzTaxon–e revealed that strain M15T was most closely related to the members of the genus Rhizobium, and showing the highest 16S rRNA gene sequence similarity to R. radiobacter LMG140T (99.64%) and R. pusense NRCPB10T (99.36%). A phylogenetic tree was constructed with the Neighbour–joining (NJ) method using MEGA 7.0 (Figure 1), which indicating the isolate was clustered into a novel branch within the genus Rhizobium.
To further explore the phylogenetic relationships between strain M15T and the closely related Rhizobium, the partial recA (496 bp), ropB (961 bp) and atpD (489 bp) gene sequences were also detected. The strain M15T shared the highest recA (91.94%), ropB (94.81%)and atpD (97.05%) gene sequences similarity with closely related Rhizobium species, so the high similarity of the housekeeping gene sequences, and their phylogenetic trees between strain M15T and closely related strains of the genus Rhizobium (Supplementary Figure S2), also indicating that strain M15T represent a novel species within the genus Rhizobium.

3.3. Cellular Fatty Acid Composition

The principal cellular fatty acids of strain M15T are Summed Feature 8 (C18:1ω7c and/or C18:1ω6c) and Summed feature 2 (aldehyde–C12:0 and/or unknown equivalent chain length), which are the characteristic compositions of root-nodule bacteria [46]. However, the novel isolate could be divided from related species mainly by possessing C9:0 and distinctly different content of the C19:0 cycloω8c. The details of the fatty acid profiles of strain M15T and reference strains are shown in Table 2.

3.4. Analysis of Plant Growth-Promoting Characteristic

To determine the properties of plant growth-promoting of strain M15T, several related functions were characterized. The results showed that strain M15T produced IAA with 85.32 μg per ml under the OD600 nm of 1.0. IAA is the main auxin in plants, regulating growth and developmental processes such as cell division and elongation, tissue differentiation, apical dominance, and responses to light, gravity, and pathogens [47]. After growth in CAS medium, we observed an orange halo surrounded by the colony indicative of siderophore production. Siderophores are low molecular-weight iron chelators that directly promote plant growth by converting unavailable ferric oxides into absorbable forms for plants or indirectly by binding to the available forms of iron in soil and depriving pathogens in the vicinity of iron [35,48]. The strain M15T as IAA and siderophore-producing bacteria, has the potential to be developed into bio–fertilizer to enhance plant growth and induce plant resistence to pathogens.
Strain M15T is able to produce ACC deaminase with the enzyme activity of 240nmol α-ketobutyrate/h·mg (Figure 2), which is high than the ACC deaminase activity level, namely approximately 20 nmol α–ketobutyrate/h·mg, to act as a PGPR [34]. Ethylene is a gaseous phytohormone, which is involved in a broad spectrum of the plant life cycle, including seed germination, nodulation, flower senescence, fruitripening, and leaf abscission [49]. High concentration of ethylene lead to growth inhibition, chlorosis, or even death of plant [49]. ACC deaminase can modulate the level of ethylene to facilitate plant resistence under different environmental stresses, especially waterlogged conditions, and contribute to plant growth and development [49,50]. Therefore, ACC deaminase–producing bacteria, like strain M15T, can be developed into inoculants to be used as alternatives to various agrochemicals.
The results of seed inoculation assays showed that the strain M15T promoted the growth of rice compared to the controls. The length of stem and fresh weight of the inoculated seedlings was significantly increased with respect to the uninoculated seedlings. These results showed that the strain M15T promoted plant growth by affecting the development of the stem and the fresh weight of rice root.

3.5. The Comparative Genome Analyses from 12 Closely Related Strains of the Genus Rhizobium

Pan–genomic analysis of the 12 Rhizobium genomes revealed that 10,182 ortholog clusters that constituted the pan-genomes. The numbers of core genomes, strain-specific genomes and accessory genomes were 2006, 8082 and 7994, respectively (Figure 3A). The number of strain–specific genes in the 12 Rhizobium species was shown in Figure 3B. The phylogenomic analysis also supported the position of the strain M15T was closest to R. radiobacter LMG140T, R. pusense NRCPB10T and R. nepotum 39/7T (Figure 3C). The results of virulence factors analysis showed that strain M15T is differ from R. radiobacter LMG140T in absence of VirB/VirD4 type IV secretion system and translocated effector Beps and virB–homolog (vbh) type IV secretion system, which is reported as a major virulence determinant for causing the crown gall disease of Rhizobium radiobacter. Therefore, despite the highest similarity to the pathogenic R. radiobacter, we can exclude the pathogenicity of strain M15T.

3.6. Secondary Metabolite Clusters

Through the anti–SMASH genome analysis tool, four gene clusters of secondary metabolites have been identified in the genome of strain M15T, one terpene, one TfuA–related, one nonribosomal peptide synthetase (NRPS) and one hserlactone (Figure 4) [37]. Synteny and gene structure analysis of the four gene clusters were carried out depending on the homology and distribution of the genes in the gene clusters.

4. Conclusions

By means of biochemical, physiological and morphological characteristics, DNA–DNA hybridization and genotypic comparison of 16S rRNA, housekeeping genes and comparative genome analysis, strains M15T are proposed to represent a novel species within the genus Rhizobium for which the name Rhizobium oryzihabitans sp. nov. is proposed. The description of Rhizobium oryzihabitans sp. nov. is summarized in Appendix A. Based on the analysis of plant growth-promoting agents, this novel isolate exhibited the capacity of producing siderophore, ACC deaminase, IAA and enhancing the rice plant growth. Thus, as PGPR, strain M15T has the potential to be exploited as sustainable and environment-friendly inoculants in agriculture replacing fertilizers for enhancing plant growth.

Patents

The work reported in the manuscript has been granted a Chinese invention patent (CN201810390383).

Supplementary Materials

The following are available online at https://www.mdpi.com/2076-2607/8/4/608/s1.

Author Contributions

J.J.Z., X.Z., J.R.W., Q.G., X.X.Z. and G.S.Z. conceived and supervised the study; X.X.Z. and G.S.Z. designed the experiments; J.J.Z., X.Z. performed the experiments; J.R.W. performed the genomic analysis; J.J.Z., J.R.W. and Q.G. analysed the data and wrote the manuscript. All authors have read and agreed to the published version of the manuscript.

Funding

This research was financially supported by National Natural Science Foundation of China (NSFC No. 31670113) and National Key Research and Development Program of China (2017YFD0201401).

Acknowledgments

The authors would like to express their gratitude to Jingnan Liang (Institute of Microbiology, Chinese Academy of Sciences) for creating the transmission electron microscopes images of strain M15T.

Conflicts of Interest

The authors declare no conflict of interest.

Appendix A

Description of Rhizobium oryzihabitans sp. nov.
Rhizobium oryzihabitans (L.n. oryza, rice; L. part. adj. habitans, inhabiting, dwelling; N.L. part. adj. oryzihabitans, rice–inhabiting).
Cells are Gram-negative, aerobic, flagellate and rods. Colonies are circular and cream-white on YMA at 30 ℃. Growth occurs from 15 ℃ to 50 ℃ and the pH range for growth is 5.0–12.0. The tolerant of NaCl concentrations is up to 7.0% (w/v). Positive reactions for nitrate reduction, aesculin hydrolysis, urease and nitro-D-methyl galactose activities, and assimilation of glucose, arabinose, mannose, mannitol, N-acetyl-glucosamine, maltose, gluconate, malic acid, citric acid, phenylacetic acid are positive. Catalase and oxidase positive. The following compounds utilize as carbon sources: N-acetyl-D-galactosamine, N-acetyl--D-glucosamine, adonitol, L-arabinose, D-arabitol, D-cellobiose, D-fructose, L-fucose, D-galactose, gentiobiose, α-D-glucose, m-inositol, α-D-lactose, lactulose, maltose, D-mannitol, D-mannose, D-melibiose, β-methyl-D-glucoside, D-raffinose, L-rhamnose, D-sorbitol, sucrose, D-trehalose, turanose, xylitol, acetic Acid, D-galactonic acid lactone, D-gluconic Acid, α-keto glutaric acid, DL-lactic acid, propionic acid, quinic acid, succinic acid, D-alanine, L-alanine, L-alanyl-glycine, L-asparagine, L-aspartic acid, L-glutamic acid, L-histidine, hydroxy-L-proline, L-ornithine, L-proline, L-pyroglutamic acid, L-serine, inosine, uridine, glycerol, glucose-1-phosphate, glucose-6-phosphate. The DNA G+C content of type strain is 59.28 mol %. Major cellular fatty acids are summed feature 8 (C18:1 ω7c and/or C18:1ω6c) and and Summed feature 2 (aldehyde-C12:0 and/or unknown equivalent chain length).
The type strain, M15T (= JCM 32903T = ACCC 60121T), was isolated from the root of rice.
The GenBank/The European Bioinformatics Institute EMBL-EBI (EMBL))/DNA Data Bank of Japan (DDBJ) accession numbers for the 16S rRNA, recA, ropB and atpD gene sequences of strain M15T are MT023790, MT028481, MT028482 and MT028483, respectively. The complete genome have been deposited in GenBank under the accession numbers of SAMN14048699.

References

  1. Frank, B. Ueber die pilzsymbiose der leguminosen. Ber. Deutsch. Bot. Ges. 1889, 7, 332–346. [Google Scholar]
  2. Behrendt, U.; Kämpfer, P.; Glaeser, S.P.; Augustin, J.; Ulrich, A. Characterization of the N2O-producing soil bacterium Rhizobium azooxidifex sp. nov. Int. J. Syst. Evol. Microbiol. 2016, 66, 2354–2361. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  3. Gu, T.; Sun, L.N.; Zhang, J.; Sui, X.H.; Li, S.P. Rhizobium flavum sp. nov., a triazophos-degrading bacterium isolated from soil under the long-term application of triazophos. Int. J. Syst. Evol. Microbiol. 2014, 64, 2017–2022. [Google Scholar] [CrossRef]
  4. Zhang, X.; Li, B.; Wang, H.; Sui, X.; Ma, X.; Hong, Q.; Jiang, R. Rhizobium petrolearium sp. nov., isolated from oil-contaminated soil. Int. J. Syst. Evol. Microbiol. 2012, 62, 1871–1876. [Google Scholar] [CrossRef] [Green Version]
  5. Yoon, J.H.; Kang, S.J.; Yi, H.S.; Oh, T.K.; Ryu, C.M. Rhizobium soli sp. nov., isolated from soil. Int. J. Syst. Evol. Microbiol. 2010, 60, 1387–1393. [Google Scholar] [CrossRef] [Green Version]
  6. Sheu, S.Y.; Chen, Z.H.; Young, C.C.; Chen, W.M. Rhizobium ipomoeae sp. nov., isolated from a water convolvulus field. Int. J. Syst. Evol. Microbiol. 2016, 66, 1633–1640. [Google Scholar] [CrossRef]
  7. Liu, Y.; Wang, R.P.; Ren, C.; Lai, Q.L.; Zeng, R.Y. Rhizobium marinum sp. nov., a malachite-green tolerant bacterium isolated from the sea water. Int. J. Syst. Evol. Microbiol. 2015, 65, 4449–4454. [Google Scholar] [CrossRef]
  8. Rosenblueth, M.; Martinez-Romero, E. Rhizobium etli maize populations and their competitiveness for root colonization. Arch. Microbiol. 2004, 181, 337–344. [Google Scholar] [CrossRef]
  9. Zhao, J.-J.; Zhang, X.; Sun, L.; Zhang, R.-J.; Zhang, C.-W.; Yin, H.-Q.; Zhang, X.-X. Rhizobium oryziradicis sp. nov. isolated from rice roots. Int. J. Syst. Evol. Microbiol. 2017, 67, 963–968. [Google Scholar] [CrossRef]
  10. Zhang, X.; Sun, L.; Ma, X.; Sui, X.H.; Jiang, R. Rhizobium pseudoryzae sp. nov. isolated from the rhizosphere of rice. Int. J. Syst. Evol. Microbiol. 2011, 61, 2425–2429. [Google Scholar] [CrossRef] [Green Version]
  11. Zhang, X.-X.; Tang, X.; Sheirdil, R.A.; Sun, L.; Ma, X.-T. Rhizobium rhizoryzae sp. nov. isolated from rice roots. Int. J. Syst. Evol. Microbiol. 2014, 64, 1373–1377. [Google Scholar] [CrossRef] [PubMed]
  12. Zhang, X.X.; Gao, J.S.; Cao, Y.H.; Sheirdil, R.A.; Sheirdil, X.C.; Zhang, L. Isolation and Proposal Novel Rice Promoting Endophytic Bacteria, Rhizobium oryzicola sp. nov. Int. J. Syst. Evol. Microbiol. 2015, 65, 2931. [Google Scholar] [CrossRef]
  13. Pravin, V.; Rosazlin, A.; Tumirah, K.; Ismail, S.; Boyce, A.N. Role of Plant Growth Promoting Rhizobacteria in Agricultural Sustainability—A Review. Molecules 2016, 21, 573. [Google Scholar]
  14. Poole, P.; Ramachandran, V.; Terpolilli, J. Rhizobia: From saprophytes to endosymbionts. Nat. Rev. Microbiol. 2018, 16, 291–303. [Google Scholar] [CrossRef]
  15. Ferreira, C.M.H.; Soares, H.M.V.; Soares, E.V. Promising bacterial genera for agricultural practices: An insight on plant growth-promoting properties and microbial safety aspects. Sci. Total Environ. 2019, 682, 779–799. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  16. García-Fraile, P.; Carro, L.; Robledo, M.; Bahena, M.H.R.; Flores-Félix, J.D.; Fernández, M.T.; Mateos, P.; Rivas, R.; Igual, J.M.; Martínez-Molina, E.; et al. Rhizobium promotes non-legumes growth and quality in several production steps: Towards a biofertilization of edible raw vegetables healthy for humans. PLoS ONE 2012, 7, e38122. [Google Scholar] [CrossRef] [Green Version]
  17. Vincent, J.M. The cultivation, isolation and maintenance of rhizobia. In A Manual for the Practical Study of the Root-Nodule Bacteria; Vincent, J.M., Ed.; Blackwell Scientific: Oxford, UK, 1970; pp. 1–13. [Google Scholar]
  18. Lane, D.J. 16S/23S rRNA sequencing. In Nucleic Acid Techniques in Bacterial Systematics; Stackebrandt, E., Goodfellow, M., Eds.; Wiley: Chichester, UK, 1991; pp. 115–175. [Google Scholar]
  19. Martens, M.; Delaere, M.; Coopman, R.; De Vos, P.; Gillis, M.; Willems, A. Multilocus sequence analysis of Ensifer and related taxa. Int. J. Syst. Evol. Microbiol. 2007, 57, 489–503. [Google Scholar] [CrossRef] [Green Version]
  20. Martens, M.; Dawyndt, P.; Coopman, R.; Gillis, M.; De Vos, P.; Willems, A. Advantages of multilocus sequence analysis for taxonomic studies: A case study using 10 housekeeping genes in the genus Ensifer (including former Sinorhizobium). Int. J. Syst. Evol. Microbiol. 2008, 58, 200–214. [Google Scholar] [CrossRef] [Green Version]
  21. Kim, O.S.; Cho, Y.J.; Lee, K.; Yoon, S.H.; Kim, M.; Na, H.; Park, S.C.; Jeon, Y.S.; Lee, J.H.; Yi, H.; et al. Introducing EzTaxon-e: A prokaryotic 16S rRNA gene sequence database with phylotypes that represent uncultured species. Int. J. Syst. Evol. Microbiol. 2012, 62, 716–721. [Google Scholar] [CrossRef]
  22. Thompson, J.D.; Higgins, D.G.; Gibson, T.J. CLUSTALW: Improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 1994, 22, 4673–4680. [Google Scholar] [CrossRef] [Green Version]
  23. Kumar, S.; Stecher, G.; Tamura, K. MEGA7: Molecular Evolutionary Genetics Analysis version 7.0 for bigger datasets. Mol. Biol. Evol. 2016, 33, 1870–1874. [Google Scholar] [CrossRef] [Green Version]
  24. Saitou, N.; Nei, M. The neighbor-joining method: A new method for reconstructing phylogenetic trees. Mol. Boil. Evol. 1987, 4, 406–425. [Google Scholar]
  25. Felsenstein, J. Evolutionary trees from DNA sequences: A maximum likelihood approach. J. Mol. Evol. 1981, 17, 368–376. [Google Scholar] [CrossRef] [PubMed]
  26. Tritt, A.; Eisen, J.A.; Facciotti, M.T.; Darling, A.E. An Integrated Pipeline for de Novo Assembly of Microbial Genomes. PLoS ONE 2012, 7, 9. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  27. Bankevich, A.; Nurk, S.; Antipov, D.; Gurevich, A.A.; Dvorkin, M.; Kulikov, A.S.; Lesin, V.M.; Nikolenko, S.I.; Pham, S.; Prjibelski, A.D.; et al. SPAdes: A New Genome Assembly Algorithm and Its Applications to Single-Cell Sequencing. J. Comput. Biol. 2012, 19, 455–477. [Google Scholar] [CrossRef] [Green Version]
  28. Chin, C.-S.; Peluso, P.; Sedlazeck, F.J.; Nattestad, M.; Concepcion, G.T.; Clum, A.; Dunn, C.; Omalley, R.; Figueroa-Balderas, R.; Morales-Cruz, A.; et al. Phased diploid genome assembly with single-molecule real-time sequencing. Nat. Methods 2016, 13, 1050–1054. [Google Scholar] [CrossRef] [Green Version]
  29. Sergey, K.; Walenz, B.P.; Berlin, K.; Miller, J.R.; Bergman, N.H.; Phillippy, A.M. Canu: Scalable and accurate long-read assembly via adaptive k-mer weighting and repeat separation. Genome Res. 2012, 27, 722–736. [Google Scholar]
  30. Meier-Kolthoff, J.P.; Auch, A.F.; Klenk, H.P.; Göker, M. Genome sequence-based species delimitation with confidence intervals and improved distance functions. BMC Bioinform. 2012, 14, 1–14. [Google Scholar] [CrossRef] [Green Version]
  31. Sasser, M. Identification of Bacteria by Gas Chromatography of Cellular Fatty Acids; MIDI Inc.: Newark, DE, USA, 1990; MIDI Technical Note 101. [Google Scholar]
  32. Glickmann, E.; Dessaux, Y.A. Critical examination of the specificity of the Salkowski reagent for indolic compounds produced by phytopathogenic bacteria. Appl. Environ. Microbiol. 1995, 619, 793–796. [Google Scholar] [CrossRef] [Green Version]
  33. Schwyn, B.; Neilands, J.B. Universal chemical assay for detection and determination of siderophore. Anal. Biochem. 1987, 160, 47–56. [Google Scholar] [CrossRef]
  34. Penrose, D.M.; Glick, B.R. Methods for isolating and characterizing ACC deaminase-containing plant growth-promoting rhizobacteria. Physiol. Plant. 2003, 118, 10–15. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  35. Habibi, S.; Djedidi, S.; Ohkama-Ohtsu, N.; Sarhadi, W.A.; Kojima, K.; Rallos, R.V.; Ramirez, M.D.A.; Yamaya, H.; Sekimoto, H.; Yokoyama, T. Isolation and Screening of Indigenous Plant Growth-promoting Rhizobacteria from Different Rice Cultivars in Afghanistan Soils. Microbes Environ. 2019, 34, 347–355. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  36. Arkin, A.P. KBase: The United States department of energy systems biology knowledgebase. Nat. Biotechnol. 2018, 36, 566–569. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  37. Price, M.N.; Dehal, P.S.; Arkin, A.P. FastTree 2-approximately maximum-likelihood trees for large alignments. PLoS ONE 2010, 5, e9490. [Google Scholar] [CrossRef]
  38. Barton, I.S.; Fuqua, C.; Platt, T.G. Ecological and evolutionary dynamics of a model facultative pathogen: Agrobacterium and crown gall disease of plants. Environ Microbiol. 2018, 20, 16–29. [Google Scholar] [CrossRef] [Green Version]
  39. Blin, K.; Shaw, S.; Steinke, K.; Villebro, R.; Ziemert, N.; Lee, S.Y.; Medema, M.H.; Weber, T. antiSMASH 5.0: Updates to the secondary metabolite genome mining pipeline. Nucleic Acids Res. 2019, 47, W81–W87. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  40. Sullivan, M.J.; Petty, N.K.; Beatson, S.A. Easyfig: A genome comparison visualizer. Bioinformatics 2011, 27, 1009–1010. [Google Scholar] [CrossRef]
  41. Jordan, D.C.; Genus, I. Rhizobium Frank 1889, 338AL. In Bergey’s Manual of Systematic Bacteriology; Krieg, N.R., Holt, J.G., Eds.; The Williams and Wilkins Co.: Baltimore, MD, USA, 1984; Volume 1, pp. 235–242. [Google Scholar]
  42. Kim, M.; Oh, H.S.; Park, S.C.; Chun, J. Towards a taxonomic coherence between average nucleotide identity and 16S rRNA gene sequence similarity for species demarcation of prokaryotes. Int. J. Syst. Evol. Microbiol. 2014, 64, 346–351. [Google Scholar] [CrossRef]
  43. Richter, M.; Rosselló-Móra, R. Shifting the genomic gold standard for the prokaryotic species definition. Proc. Natl. Acad. Sci. USA 2009, 106, 19126–19131. [Google Scholar] [CrossRef] [Green Version]
  44. Graham, P.H.; Sadowsky, M.J.; Keyser, H.H.; Barnet, Y.M.; Bradley, R.S. Proposed minimal standards for the description of new genera and species of root- and stem-nodulating bacteria. Int. J. Syst. Bacteriol. 1991, 41, 582–587. [Google Scholar] [CrossRef] [Green Version]
  45. Wayne, L.G.; Brenner, D.J.; Colwell, R.R.; Grimont, P.A.D.; Kandler, O. International Committee on Systematic Bacteriology. Report of the ad hoc committee on reconciliation of approaches to bacterial systematics. Int. J. Syst. Bacteriol. 1987, 37, 463–464. [Google Scholar] [CrossRef] [Green Version]
  46. Tighe, S.W.; De Lajudie, P.; DiPietro, K.; Lindström, K.; Nick, G.; Jarvis, B.D. Analysis of cellular fatty acids and phenotypic relationships of Agrobacterium, Bradyrhizobium, Mesorhizobium, Rhizobium and Sinorhizobium species using the Sherlock Microbial Identification System. Int. J. Syst. Evol. Microbiol. 2000, 50, 787–801. [Google Scholar] [CrossRef] [PubMed]
  47. Fu, S.F.; Wei, J.Y.; Chen, H.W.; Liu, Y.Y.; Lu, H.Y.; Chou, J.Y. Indole-3-acetic acid: A widespread physiological code in interactions of fungi with other organisms. Plant Signal. Behav. 2015, 10, e1048052. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  48. Marques, A.P.G.C.; Pires, C.; Moreira, H.; Ragel, A.O.S.S.; Castro, P.M.L. Assessment of the plant growth promotion abilities of six bacterial isolates using Zea mays as indicator plant. Soil Biol. Biochem. 2010, 42, 1229–1235. [Google Scholar] [CrossRef] [Green Version]
  49. Ali, S.; Kim, W.C. Plant Growth Promotion under Water: Decrease of Waterlogging-Induced ACC and Ethylene Levels by ACC Deaminase-Producing Bacteria. Front Microbiol. 2018, 9, 1096. [Google Scholar] [CrossRef] [PubMed]
  50. Tavares, M.J.; Nascimento, F.X.; Glick, B.R.; Rossi, M.J. The expression of an exogenous ACC deaminase by the endophyte Serratia grimesii BXF1 promotes the early nodulation and growth of common bean. Lett. Appl. Microbiol. 2018, 66, 252–259. [Google Scholar] [CrossRef]
Microorganisms 08 00608 g001 550
Figure 1. Phylogenetic tree generated with the neighbor-joining algorithm based on 16S rRNA gene sequences showing the phylogenetic positions of strain M15T and related taxa. Bootstrap values with more than 50% are shown on the nodes as percentages of 1000 replicates. Rhizobium grahamii CCGE502T (AEYE01000061) was used as an outgroup. The scale bar equals 0.005 change per nucleotide position.
Figure 1. Phylogenetic tree generated with the neighbor-joining algorithm based on 16S rRNA gene sequences showing the phylogenetic positions of strain M15T and related taxa. Bootstrap values with more than 50% are shown on the nodes as percentages of 1000 replicates. Rhizobium grahamii CCGE502T (AEYE01000061) was used as an outgroup. The scale bar equals 0.005 change per nucleotide position.
Microorganisms 08 00608 g001
Microorganisms 08 00608 g002 550
Figure 2. (a) Phosphate solubilization test in PKO medium; (b) detection of indole acetic acid production; CK− indicates the negative control using non-inoculated medium mixed with an equal volume of colorimetric; CK+ indicates the positive control using 100 μg/ml indole-3-acetic acid (IAA) standard mixed with an equal volume of colorimetric; (c) siderophore production with three repetitions; (d,e) Effects of strain M15T on rice plant growth; d, e, indicate the effects of strain M15T on rice stem length and fresh weight (total weight of roots and stems), respectively. ** value is significantly different from the control, within each column (P < 0.01); * value is significantly different from the control, within each column (P < 0.05); Each treatment have at least three biological replicates.
Figure 2. (a) Phosphate solubilization test in PKO medium; (b) detection of indole acetic acid production; CK− indicates the negative control using non-inoculated medium mixed with an equal volume of colorimetric; CK+ indicates the positive control using 100 μg/ml indole-3-acetic acid (IAA) standard mixed with an equal volume of colorimetric; (c) siderophore production with three repetitions; (d,e) Effects of strain M15T on rice plant growth; d, e, indicate the effects of strain M15T on rice stem length and fresh weight (total weight of roots and stems), respectively. ** value is significantly different from the control, within each column (P < 0.01); * value is significantly different from the control, within each column (P < 0.05); Each treatment have at least three biological replicates.
Microorganisms 08 00608 g002
Microorganisms 08 00608 g003a 550Microorganisms 08 00608 g003b 550
Figure 3. The comparative genome analysis of 12 closely related Rhizobium genomes. (a) The phylogenetic tree with the insert genome into species. (b) Genomic diversity numerically showing the homologous and non-homologous genes. Each strain was represented by an oval. Shared genes and species–specific genes were shown in center circle and petals, respectively. The strain name was located beside the oval. (c) Core genome-based phylogeny based on 2006 core orthologous proteins of strain M15T and closely related species of genus Rhizobium.
Figure 3. The comparative genome analysis of 12 closely related Rhizobium genomes. (a) The phylogenetic tree with the insert genome into species. (b) Genomic diversity numerically showing the homologous and non-homologous genes. Each strain was represented by an oval. Shared genes and species–specific genes were shown in center circle and petals, respectively. The strain name was located beside the oval. (c) Core genome-based phylogeny based on 2006 core orthologous proteins of strain M15T and closely related species of genus Rhizobium.
Microorganisms 08 00608 g003aMicroorganisms 08 00608 g003b
Microorganisms 08 00608 g004 550
Figure 4. Comparison of biosynthetic gene clusters between strain M15T and other closely related type species of the genus Rhizobium. Regions of conserved synteny were marked with grey (+) and green (−) shadow. Different genes are shown by different color arrows, and genes with the same color are homologous to each other.
Figure 4. Comparison of biosynthetic gene clusters between strain M15T and other closely related type species of the genus Rhizobium. Regions of conserved synteny were marked with grey (+) and green (−) shadow. Different genes are shown by different color arrows, and genes with the same color are homologous to each other.
Microorganisms 08 00608 g004
Table
Table 1. The phenotypic characteristics of strain M15T and closely related strains. 1, M15T; 2, R. radiobacter LMG140T; 3, R. pusense NRCPB10T. All data are obtained from the present study. + (positive); w (weakly positive); − (negative).
Table 1. The phenotypic characteristics of strain M15T and closely related strains. 1, M15T; 2, R. radiobacter LMG140T; 3, R. pusense NRCPB10T. All data are obtained from the present study. + (positive); w (weakly positive); − (negative).
Characteristics123
Growth in/at
NaCl (7.0%) +--
pH 11+-+
Carbon-source utilization
Dextrin -ww
N-acetyl-D-galactosamine ++-
Lactulose w++
D-psicose -++
Methyl pyruvate -+w
Mono-methyl-succinate-+w
Formic acid -w+
β-hydroxy butyric acid -+w
α-keto glutaric acid ++-
L-alanine ++w
L-threonine -ww
Inosine w--
Uridinew--
2,3-Butanediol --w
Table
Table 2. Cellular fatty acid contents of strain M15T and the type strains of related Rhizobium type species. 1, M15T; 2, R. radiobacter LMG140T; 3, R. pusense NRCPB10T. All data were obtained in this study. Fatty acids representing < 1% in all strains were omitted. -, not detected.
Table 2. Cellular fatty acid contents of strain M15T and the type strains of related Rhizobium type species. 1, M15T; 2, R. radiobacter LMG140T; 3, R. pusense NRCPB10T. All data were obtained in this study. Fatty acids representing < 1% in all strains were omitted. -, not detected.
Fatty Acid123
C9:01.03--
C13:1 at 12–133.21.751.98
C16:02.166.6210.77
C16:0 3-OH1.885.385.47
C19:0 cycloω8c3.1338.2340.77
Summed Feature 2 *38.5411.93-
Summed Feature 3 *1.651.032.22
Summed Feature 8 *40.9926.5430.94
Note: * Summed features consist of two or more fatty acids that could not be separated by the Microbial Identification System. Summed feature 2 comprised aldehyde-C12: 0 and/or unknown equivalent chain length (ECL) 10.9525; summed feature 3 comprised C16: 1ω7c and/or C16: 1ω6c; summed feature 8 comprised C18: 1ω7c and/or C18: 1ω6c.

Share and Cite

MDPI and ACS Style

Zhao, J.; Zhao, X.; Wang, J.; Gong, Q.; Zhang, X.; Zhang, G. Isolation, Identification and Characterization of Endophytic Bacterium Rhizobium oryzihabitans sp. nov., from Rice Root with Biotechnological Potential in Agriculture. Microorganisms 2020, 8, 608. https://doi.org/10.3390/microorganisms8040608

AMA Style

Zhao J, Zhao X, Wang J, Gong Q, Zhang X, Zhang G. Isolation, Identification and Characterization of Endophytic Bacterium Rhizobium oryzihabitans sp. nov., from Rice Root with Biotechnological Potential in Agriculture. Microorganisms. 2020; 8(4):608. https://doi.org/10.3390/microorganisms8040608

Chicago/Turabian Style

Zhao, Juanjuan, Xia Zhao, Junru Wang, Qi Gong, Xiaoxia Zhang, and Guishan Zhang. 2020. "Isolation, Identification and Characterization of Endophytic Bacterium Rhizobium oryzihabitans sp. nov., from Rice Root with Biotechnological Potential in Agriculture" Microorganisms 8, no. 4: 608. https://doi.org/10.3390/microorganisms8040608

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Metrics

Back to TopTop