Abstract
Certain strains of Bacillus amyloliquefaciens can colonize plants and improve growth and stress management. In order to study these effects, bacterial growth dynamics on plants and in the rhizosphere are of interest calling for specific analytical tools. For that purpose, quantitative real-time PCR (qPCR) assays were developed in order to differentiate among three closely related B. amyloliquefaciens subsp. plantarum strains (UCMB5033, UCMB5036, UCMB5113) and to determine their levels with high accuracy. Oligonucleotide primers were designed for strain unique gene sequences and used for SYBR green based qPCR analysis. Standard curves covered a wide linear range (106) of DNA amounts with the lowest detection level at 50 fg. Post-reaction melting curve analysis showed only a single product. Accurate threshold cycles were obtained, even in the presence of high excess of related Bacillus strains and total bacterial DNA from soil. Analysis of Bacillus colonisation after seed treatment of two oilseed rape cultivars (Oase and Ritz) grown on agar support showed a time dependent effect but that the bacteria mostly were found on root tissues and little on green tissues. The colonisation on plants grown in soil varied among the Bacillus strains where Oase seemed to house more bacteria than Ritz. Applied as a mixture, all three Bacillus strains co-existed on the roots of plants grown in soil. The qPCR assay in combination with other techniques will be a powerful tool to study plant interactions of these B. amyloliquefaciens biocontrol agents to further understand the requirements for successful interactions and improvement of plant properties.
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Abbreviations
- DAI:
-
Days after inoculation
- PGPB:
-
Plant growth-promoting bacteria
- qPCR:
-
Quantitative real-time PCR
References
Andrews M, Cripps MG, Edwards GR (2012) The potential of beneficial microorganisms in agricultural systems. Ann Appl Biol 160:1–5
Bais HP, Weir TL, Perry LG, Gilroy S, Vivanco JM (2006) The role of root exudates in rhizosphere interactions with plants and other organisms. Annu Rev Plant Biol 57:233–266
Bejai SR, Danielsson J, Meijer J (2009) Transcript profiling of oilseed rape (Brassica napus) primed for biocontrol differentiate genes involved in microbial interactions with beneficial Bacillus amyloliquefaciens from pathogenic Botrytis cinerea. Plant Mol Biol 70:31–45
Bhattacharyya PN, Jha DK (2012) Plant growth-promoting rhizobacteria (PGPR): emergence in agriculture. World J Microbiol Biotechnol 28:1327–1350
Bogino PC, Oliva Mde L, Sorroche FG, Giordano W (2013) The role of bacterial biofilms and surface components in plant-bacterial associations. Int J Mol Sci 14:15838–15859
Compant S, Clement C, Sessitsch A (2010) Plant growth-promoting bacteria in the rhizo- and endosphere of plants: their role, colonization, mechanisms involved and prospects for utilization. Soil Biol Biochem 42:669–678
Curtis TP, Sloan WT (2005) Exploring microbial diversity- a vast below. Science 309:1331–1333
Danielsson J, Reva O, Meijer J (2007) Protection of oilseed rape (Brassica napus) toward fungal pathogens by strains of plant-associated Bacillus amyloliquefaciens. Microb Ecol 54:134–140
Duca D, Lorv J, Patten CL, Rose D, Glick BR (2014) Indole-3-acetic acid in plant–microbe interactions. Antonie Van Leeuwenhoek 106:85–125
Fritsche-Neto R, Borém A (2012) Plant breeding for biotic stress resistance. Springer-Verlag, Berlin
Gans J, Wolinsky M, Dunbar J (2005) Computational improvements reveal great bacterial diversity and high metal toxicity in soil. Science 309:1387–1390
Hawes MC, Curlango-Rivera G, Xiong Z, Kessler JO (2012) Roles of root border cells in plant defense and regulation of rhizosphere microbial populations by extracellular DNA ‘trapping’. Plant Soil 355:1–16
Hirsch PR, Mauchline TH, Clark IM (2010) Culture-independent molecular techniques for soil microbial ecology. Soil Biol Biochem 42:878–887
Lakshmanan V, Kitto SL, Caplan JL, Hsueh YH, Kearns DB, Wu YS, Bais HP (2012) Microbe-associated molecular patterns-triggered root responses mediate beneficial rhizobacterial recruitment in Arabidopsis. Plant Physiol 160:1642–1661
Lombard N, Prestat E, van Elsas JD, Simonet P (2011) Soil-specific limitations for access and analysis of soil microbial communities by metagenomics. FEMS Microbiol Ecol 78:31–49
Manzoor S, Niazi A, Bejai S, Meijer J, Bongcam-Rudloff E (2013) Genome sequence of a plant-associated bacterium, Bacillus amyloliquefaciens strain UCMB5036. Genome Announc 1(2):e0011113
McSpadden Gardener B (2004) Ecology of Bacillus and Paenibacillus spp. in agricultural systems. Phytopathology 94:1252–1258
Mercado-Blanco J, Bakker PAHM (2007) Interactions between plants and beneficial Pseudomonas spp.: exploiting bacterial traits for crop protection. Antonie Van Leeuwenhoek 92:367–389
Meyer JB, Lutz MP, Frapolli M, Péchy-Tarr M, Rochat L, Keel C, Défago G, Maurhofer M (2010) Interplay between wheat cultivars, biocontrol Pseudomonads, and soil. Appl Environ Microbiol 76:6196–6204
Niazi A, Manzoor S, Bejai S, Meijer J, Bongcam-Rudloff E (2014a) Complete genome sequence of a plant associated bacterium Bacillus amyloliquefaciens strain UCMB5033. Standards in Genomic Sciences 9, No 3. doi:10.4056/sigs.4758653
Niazi A, Manzoor S, Asari S, Bejai S, Meijer J, Bongcam-Rudloff E (2014b) Genome analysis of Bacillus amyloliquefaciens subsp. plantarum UCMB5113: a rhizobacterium that improves plant growth and stress management. PLoS ONE 9(8):e104651. doi:10.1371/journal.pone.0104651
Nicholson WL (2002) Roles of Bacillus endospores in the environment. Cell Mol Life Sci 59:410–416
Philippot L, Ritz K, Pandard P, Hallin S, Martin-Laurent F (2012) Standardisation of methods in soil microbiology: progress and challenges. FEMS Microbiol Ecol 82:1–10
Raaijmakers JM, Mazzola M (2012) Diversity and natural functions of antibiotics produced by beneficial and plant pathogenic bacteria. Annu Rev Phytopathol 50:403–424
Reva ON, Dixelius C, Meijer J, Priest FG (2004) Taxonomic characterization and plant colonizing abilities of some bacteria related to Bacillus amyloliquefaciens and Bacillus subtilis. FEMS Microbiol Ecol 48:249–259
Rudrappa T, Czymmek KJ, Paré PW, Bais HP (2008) Root-secreted malic acid recruits beneficial soil bacteria. Plant Physiol 148:1547–1556
Tikhonovich IA, Provorov NA (2011) Microbiology is the basis of sustainable agriculture: an opinion. Ann Appl Biol 159:155–168
van Hulten M, Pelser M, van Loon LC, Pieterse CMJ, Ton J (2006) Costs and benefits of priming for defense in Arabidopsis. Proc Natl Acad Sci USA 103:5602–5607
van Loon LC, Bakker PAHM, Pieterse CMJ (1998) Systemic resistance induced by rhizosphere bacteria. Annu Rev Phytopathol 36:453–483
Yang J, Kloepper JW, Ryu CM (2009) Rhizosphere bacteria help plants tolerate abiotic stress. Trends Plant Sci 14:1–4
Acknowledgments
We would like to thank Ingrid Eriksson and Urban Pettersson for technical support and the SNP&SEQ Technology Platform of the Genomics platform of Science for Life Laboratory in Uppsala for Bacillus DNA sequence analysis. These studies were supported by the Swedish Research Council for Environment, Agricultural Sciences and Spatial Planning (FORMAS), Carl Tryggers Stiftelse, the Helge Ax:son Johnsons fond, the Nilsson-Ehle fund and the Higher Education Commission of Pakistan. Funding for plant growth facilities were provided in part by KFI-VR. The authors declare that they have no conflict of interest.
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Johansson, A.H., Bejai, S., Niazi, A. et al. Studies of plant colonisation by closely related Bacillus amyloliquefaciens biocontrol agents using strain specific quantitative PCR assays. Antonie van Leeuwenhoek 106, 1247–1257 (2014). https://doi.org/10.1007/s10482-014-0295-0
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DOI: https://doi.org/10.1007/s10482-014-0295-0