Aqueous peat extract exposes rhizobia to sub-lethal stress which may prime cells for improved desiccation tolerance
Inoculation of legume seed with rhizobia is an efficient and cost-effective means of distributing elite rhizobial strains to broad-acre crops and pastures. However, necessary drying steps after coating seed expose rhizobia to desiccation stress reducing survival and limiting potential nitrogen fixation by legumes. Rhizobial tolerance to desiccation varies with strain and with growth conditions prior to drying. Cells grown in peat generally survive desiccation better than cells grown in liquid broth. We aimed to identify peat-induced proteomic changes in rhizobia that may be linked to desiccation tolerance. Proteins expressed differentially after growth in peat extract when compared with a minimal defined medium were measured in four rhizobial strains. Proteins showing the greatest increase in abundance were those involved in amino acid and carbohydrate transport and metabolism. Proteins involved in posttranslational modification and cell defence mechanisms were also upregulated. Many of the proteins identified in this study have been previously linked to stress responses. In addition, analysis using nucleic acid stains SYTO9 and propidium iodide indicated that membranes had been compromised after growth in peat extract. We targeted the membrane repair protein PspA (ΔRL3579) which was upregulated in Rhizobium leguminosarum bv. viceae 3841 after growth in peat extract to validate whether the inability to repair membrane damage after growth in peat extract reduced desiccation tolerance. The ΔRL3579 mutant grown in peat extract had significantly lower survival under desiccation stress, whereas no difference in survival between wild-type and mutant strains was observed after growth in tryptone yeast (TY) or minimal medium (JMM) media. Staining mutant and wild-type strains with SYTO9 and propidium iodide indicated that membranes of the mutant were compromised after growth in peat extract and to a lesser extent in TY. This study shows that growth in peat extract causes damage to cell membranes and exposes rhizobia to sub-lethal stress resulting in differential expression of several stress-induced proteins. The induction of these proteins may prime and protect the cells when subjected to subsequent stress such as desiccation. Identifying the key proteins involved in desiccation tolerance and properties of peat that stimulate this response will be important to inform development of new inoculant technology that maximises survival of rhizobia during delivery to legume crops and pastures.
KeywordsRhizobia Inoculant technology Desiccation tolerance Peat
We acknowledge the facilities, as well as the scientific and technical assistance from the Mass Spectrometry Core Facility at the University of Sydney.
This study was funded by the Australia Awards and Grains Research and Development Corporation (GRS135 and US00065) through the University of Sydney.
Compliance with ethical standards
Conflict of interest
The authors declare that they have no conflict of interest.
This article does not contain any studies with human participants or animals performed by any of the authors.
- Balestrino D, Ghigo JM, Charbonnel N, Haagensen JA, Forestier C (2008) The characterization of functions involved in the establishment and maturation of Klebsiella pneumoniae in vitro biofilm reveals dual roles for surface exopolysaccharides. Environ Microbiol 10(3):685–701CrossRefPubMedGoogle Scholar
- Dart P, Roughley R, Chandler MR (1969) Peat culture of Rhizobium trifolii: an examination by electron microscopy. J Appl Microbiol 32(3):352–357Google Scholar
- Davey HM, Hexley P (2011) Red but not dead? Membranes of stressed Saccharomyces cerevisiae are permeable to propidium iodide. 13:163–171. https://doi.org/10.1111/j.1462-2920.2010.02317.x
- Deaker R, Hartley E, Gemell G (2012) Conditions affecting shelf-life of inoculated legume seed. Agric J 2(1):38–51Google Scholar
- Glenn R, Poole PS, Hudman JF (1980) SHORT COMMUNICATION Succinate Uptake by Free-living and Bacteroid Forms of Rhizobium leguminosarum. J Gen Microbiol 119:267–271Google Scholar
- Kedzierska S, Matuszewska E (2001) The effect of co-overproduction of DnaK/DnaJ/GrpE and ClpB proteins on the removal of heat-aggregated proteins from Escherichia coli ΔclpB mutant cells—new insight into the role of Hsp70 in a functional cooperation with Hsp100. FEMS Microbiol Lett 204(2):355–360PubMedGoogle Scholar
- Lesueur D, Deaker R, Herrmann L, Bräu L, Jansa J (2016) The production and potential of biofertilizers to improve crop yields. In: Arora NK, Mehnaz S, Balestrini R (eds) Bioformulations: for sustainable agriculture. Springer, New Delhi, pp 71–92Google Scholar
- O’Brien KM, Dirmeier R, Engle M, Poyton RO (2004) Mitochondrial protein oxidation in yeast mutants lacking manganese-(MnSOD) or copper-and zinc-containing superoxide dismutase (CuZnSOD): evidence that MnSOD AND CuZnSOD have both unique and overlapping functions in protecting mitochondrial proteins from oxidative damage. J Biol Chem 279(50):51817–51827CrossRefPubMedGoogle Scholar
- Russo DM, Williams A, Edwards A, Posadas DM, Finnie C, Dankert M, Downie JA, Zorreguieta A (2006) Proteins exported via the PrsD-PrsE type I secretion system and the acidic exopolysaccharide are involved in biofilm formation by Rhizobium leguminosarum. J Bacteriol 188(12):4474–4486CrossRefPubMedPubMedCentralGoogle Scholar
- Sambrook J, Fritsch E, Maniatis T (1989) Molecular cloning: a laboratory manual, 2nd edn. Cold Spring Harbor Press, New YorkGoogle Scholar
- Simonin H, Beney L, Gervais P (2007) Sequence of occurring damages in yeast plasma membrane during dehydration and rehydration : Mechanisms of cell death. i:1600–1610. https://doi.org/10.1016/j.bbamem.2007.03.017
- Siqueira AF, Ormeño-Orrillo E, Souza RC, Rodrigues EP, Almeida LGP, Barcellos FG, Batista JSS, Nakatani AS, Martínez-Romero E, Vasconcelos ATR, Hungria M (2014) Comparative genomics of Bradyrhizobium japonicum CPAC 15 and Bradyrhizobium diazoefficiens CPAC 7: elite model strains for understanding symbiotic performance with soybean. BMC Genomics 15(1):420CrossRefPubMedPubMedCentralGoogle Scholar
- Vanderlinde EM, Muszyński A, Harrison JJ, Koval SF, Foreman DL, Ceri H, Kannenberg EL, Carlson RW, Yost CK (2009) Rhizobium leguminosarum biovar viciae 3841, deficient in 27-hydroxyoctacosanoate-modified lipopolysaccharide, is impaired indesiccation tolerance, biofilm formation and motility. Microbiology 155:3055–3069. https://doi.org/10.1099/mic.0.025031-0 CrossRefPubMedPubMedCentralGoogle Scholar
- Vanderlinde EM, Magnus SA, Tambalo DD, Koval SF, Yost CK (2011) Mutation of a broadly conserved operon (RL3499-RL3502) from Rhizobium leguminosarum biovar viciae causes defects in cell morphology and envelope integrity. J Bacteriol 193:2684–2694. https://doi.org/10.1128/JB.01456-10 CrossRefPubMedPubMedCentralGoogle Scholar
- Vizcaino JA, Cote RG, Csordas A, Dianes JA, Fabregat A, Foster JM, Griss J, Alpi E, Birim M, Contell J, O’Kelly G, Schoenegger A, Ovelleiro D, Perez-Riverol Y, Reisinger F, Rios D, Wang R, Hermjakob H (2013) The Proteomics Identifications (PRIDE) database and associated tools: status in 2013. Nucleic Acids Res 41(D1):D1063–D1069. https://doi.org/10.1093/nar/gks1262 CrossRefPubMedGoogle Scholar