Studies of plant colonisation by closely related Bacillus amyloliquefaciens biocontrol agents using strain specific quantitative PCR assays
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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.
KeywordsColonisation Bacillus amyloliquefaciens Brassica napus Rhizosphere Beneficial bacteria Biocontrol
Days after inoculation
Plant growth-promoting bacteria
Quantitative real-time PCR
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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