Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2017 Jul 14;8:1268.
doi: 10.3389/fmicb.2017.01268. eCollection 2017.

Genetic Diversity of Nitrogen-Fixing and Plant Growth Promoting Pseudomonas Species Isolated From Sugarcane Rhizosphere

Affiliations
Free PMC article

Genetic Diversity of Nitrogen-Fixing and Plant Growth Promoting Pseudomonas Species Isolated From Sugarcane Rhizosphere

Hai-Bi Li et al. Front Microbiol. .
Free PMC article

Abstract

The study was designed to isolate and characterize Pseudomonas spp. from sugarcane rhizosphere, and to evaluate their plant- growth- promoting (PGP) traits and nitrogenase activity. A biological nitrogen-fixing microbe has great potential to replace chemical fertilizers and be used as a targeted biofertilizer in a plant. A total of 100 isolates from sugarcane rhizosphere, belonging to different species, were isolated; from these, 30 isolates were selected on the basis of preliminary screening, for in vitro antagonistic activities against sugarcane pathogens and for various PGP traits, as well as nitrogenase activity. The production of IAA varied from 312.07 to 13.12 μg mL-1 in tryptophan supplemented medium, with higher production in AN15 and lower in CN20 strain. The estimation of ACC deaminase activity, strains CY4 and BA2 produced maximum and minimum activity of 77.0 and 15.13 μmoL mg-1 h-1. For nitrogenase activity among the studied strains, CoA6 fixed higher and AY1 fixed lower in amounts (108.30 and 6.16 μmoL C2H2 h-1 mL-1). All the strains were identified on the basis of 16S rRNA gene sequencing, and the phylogenetic diversity of the strains was analyzed. The results identified all strains as being similar to Pseudomonas spp. Polymerase chain reaction (PCR) amplification of nifH and antibiotic genes was suggestive that the amplified strains had the capability to fix nitrogen and possessed biocontrol activities. Genotypic comparisons of the strains were determined by BOX, ERIC, and REP PCR profile analysis. Out of all the screened isolates, CY4 (Pseudomonas koreensis) and CN11 (Pseudomonas entomophila) showed the most prominent PGP traits, as well as nitrogenase activity. Therefore, only these two strains were selected for further studies; Biolog profiling; colonization through green fluorescent protein (GFP)-tagged bacteria; and nifH gene expression using quantitative real-time polymerase chain reaction (qRT-PCR) analysis. The Biolog phenotypic profiling, which comprised utilization of C and N sources, and tolerance to osmolytes and pH, revealed the metabolic versatility of the selected strains. The colonization ability of the selected strains was evaluated by genetically tagging them with a constitutively expressing GFP-pPROBE-pTetr-OT plasmid. qRT-PCR results showed that both strains had the ability to express the nifH gene at 90 and 120 days, as compared to a control, in both sugarcane varieties GT11 and GXB9. Therefore, our isolated strains, P. koreensis and P. entomophila may be used as inoculums or in biofertilizer production for enhancing growth and nutrients, as well as for improving nitrogen levels, in sugarcane and other crops. The present study, to the best of our knowledge, is the first report on the diversity of Pseudomonas spp. associated with sugarcane in Guangxi, China.

Keywords: Biolog; GFP; Pseudomonas; antibiotic gene; genetic diversity; nifH; sugarcane.

Figures

Figure 1
Figure 1
The 16S rDNA phylogenetic tree of Pseudomonas isolates from sugarcane. The evolutionary history was inferred using the UPGMA method. Bootstrap values of 1,000 replications are indicated as percent confidence values for particular branching. All positions containing gaps and missing data were eliminated. Sequences indicated in code were determined in this study. The E. coli was used as an out group.
Figure 2
Figure 2
PCR-amplification of nifH gene from genomic DNA of Pseudomanas species. M, molecular size marker from 100 to 2,000 bp. P is a positive control (Klebsiella verticola- DX120E) and N is a negative control (Water). M, molecular size marker (100 bp–2 kb), (Takara).
Figure 3
Figure 3
PCR finger printing patterns from genomic DNA of Pseudomonas strains isolated from sugarcane. The BOX, ERIC and REP patterns are shown in (A–C), respectively. B1-B30 (BOX), E1-E30 (ERIC), and R1-R30 (REP) is a strain codes used in this study. A 12–15 μL of each product is loaded onto a 1.5% agarose gel. M, molecular size marker (100 bp–5 kb), low range DNA ruler (Takara). Strain codes: 1. CoY1, 2. CoA5, 3. CoA6, 4. AY1, 5. AY2, 6. AA8, 7. AN15, 8. BY2, 9. BY3, 10. BY10, 11. BA2, 12. BA4, 13. BA7, 14. BA12, 15. BA13, 16. BA17, 17. BN6, 18. BN7, 19. CY4, 20. CY7, 21. CA5, 22. CA7, 23. CN1, 24. CN2, 25. CN3, 26. CN9, 27. CN11, 28. CN12, 29. CN15, and 30. CN20.
Figure 4
Figure 4
Cluster analyses of (A) BOX, (B) ERIC, and (C) REP-PCR fingerprints showing the genotypic diversity of Pseudomonas species isolated from sugarcane. Dendrogram was obtained from the similarity coefficient calculations and clustering was done using unweighted pair-grouping method based on arithmetic averages (UPGMA) using NTSYS software and the Jaccard coefficient.
Figure 5
Figure 5
Metabolic differences among CY4 and CN11 in presence of sugars, chemical sensivity, acidic pH, sodium chloride, lactic acid, hexose PO4, amino acid, hexose acid, reducing sugar, and carboxylic acid using BIOLOG Phenotype Micro-Array™ plates GNIII.
Figure 6
Figure 6
The scatterplots of selected two strains Pseudomonas koreensis (CY4) and Pseudomonas entomophila (CN11) were analyzed through principle component analysis (PCA) under the different treatments by using the BIOLOG(R) micro-plates (A) nitrogen, (B) osmolytes, and (C) pH.
Figure 7
Figure 7
Confocal laser scanning micrographs images showing gfp-tagged strains (CY4 and CN11) colonized in and on roots and leaves of sugarcane micropropagated plantlets GT11 (variety). (A–C) is control sugarcane plantlets parts i.e., stem, leaf and root, without inoculated strains. Confocal microscopic images (D–G) present inoculated bacterial GFP fluorescence (500–530 nm) in green dots and auto-fluorescence in everywhere in leaf and root. Arrow heads point indicates bacterial cells present in a single or grouped of bacteria. (D,E) represents CY4 and (F,G) is CN11 strain. Bars present 50 μm.
Figure 8
Figure 8
qRT-PCR analysis of the nifH expression patterns in sugarcane varieties (GT11 and GXB9) and potent strains (Pseudomonas koreensis-CY4 and Pseudomonas entomophila-CN11) interaction. Data were normalized to the GAPDH expression level. All data points (with the deduction of their controls) are the means ± SE (n = 3).

Similar articles

See all similar articles

Cited by 6 articles

See all "Cited by" articles

References

    1. Ahemad M., Khan M. S. (2012a). Effect of fungicides on plant growth promoting activities of phosphate solubilizing Pseudomonas putida isolated from mustard (Brassica compestris) rhizosphere. Chemosphere. 86, 945–950. 10.1016/j.chemosphere.2011.11.013 - DOI - PubMed
    1. Ahemad M., Khan M. S. (2012b). Evaluation of plant growth promoting activities of rhizobacterium Pseudomonas putida under herbicide-stress. Ann. Microbiol. 62, 1531–1540. 10.1007/s13213-011-0407-2 - DOI
    1. Akter Z., Pageni B. B., Lupwayi N. Z., Balasubramanian P. M. (2014). Biological nitrogen fixation and nifH gene expression in dry beans (Phaseolus vulgaris L.). Can. J. Plant Sci. 94, 203–212. 10.4141/cjps2013-200 - DOI
    1. Antwerpen T. V., Rutherford R. S., Vogel J. L. (2002). Assessment of sugarcane endophytic bacteria and rhizospheric Burkholderia species as antifungal agents. Proc. S. Afr. Sug. Technol. Assoc. 76, 301–304.
    1. Anzai Y., Kim H., Park J. Y., Wakabayashi H., Oyaizu H. (2000). Phylogenetic affiliation of the pseudomonads based on 16S rRNA sequence. Int. J. Syst. Evol. Microbiol. 4, 1563–1589. 10.1099/00207713-50-4-1563 - DOI - PubMed
Feedback