title: Development of a real-time PCR assay for detection and quantification of Rhizobium leguminosarum bacteria and discrimination between different biovars in zinc-contaminated soil.
abstract: Primers were designed to target 16S rRNA and nodD genes of Rhizobium leguminosarum from DNA extracted from two different soil types contaminated with Zn applied in sewage sludge. Numbers of rhizobia estimated using 16S rRNA gene copy number showed higher abundance than those estimated by both nodD and the most-probable-number (MPN) enumeration method using a plant trap host. Both 16S rRNA gene copies and the MPN rhizobia declined with increased levels of Zn contamination, as did the abundance of the functional gene nodD, providing compelling evidence of a toxic effect of Zn on R. leguminosarum populations in the soil. Regression analysis suggested the total Zn concentration in soil as a better predictor of rhizobial numbers than both NH4NO3-extractable and soil solution Zn. R. leguminosarum bv. viciae nodD gene copies were generally less sensitive to Zn than R. leguminosarum bv. trifolii nodD. The latter were generally below detection limits at Zn levels of >250 mg kg(-1). Although there were differences in the actual numbers estimated by each approach, the response to Zn was broadly similar across all methods. These differences were likely to result from the fact that the molecular approaches assess the potential for nodulation while the MPN approach assesses actual nodulation. The results demonstrate that the use of targeted gene probes for assessing environmental perturbations of indigenous soil rhizobial populations may be more sensitive than the conventional plant bioassay and MPN methods.
authors: Department of Soil Science, Rothamsted Research, Harpenden, Hertfordshire AL5 2JQ, United Kingdom. c.macdonald@uws.edu.au
authors: Catriona A
authors: CA
authors: Macdonald
authors: Ian M
authors: IM
authors: Clark
authors: Penny R
authors: PR
authors: Hirsch
authors: Fang-Jie
authors: FJ
authors: Zhao
authors: Steve P
authors: SP
authors: McGrath
references: Environ Microbiol. 2005 Jul;7(7):1049-58
references: 15946302
references: J Appl Microbiol. 2007 Nov;103(5):1355-65
references: 17953546
references: Antonie Van Leeuwenhoek. 2002 Aug;81(1-4):607-16
references: 12448756
references: Lett Appl Microbiol. 2009 Mar;48(3):355-61
references: 19207854
references: Environ Microbiol. 2001 Jul;3(7):431-9
references: 11553233
references: Nucleic Acids Res. 2004 Mar 19;32(5):1792-7
references: 15034147
references: Environ Pollut. 2001;111(2):311-20
references: 11202735
references: Appl Environ Microbiol. 1985 Jun;49(6):1482-7
references: 16346816
references: Microbiol Res. 2004;159(2):121-7
references: 15293945
references: Curr Microbiol. 2007 Mar;54(3):167-74
references: 17253091
references: Nucleic Acids Res. 2003 Jan 1;31(1):442-3
references: 12520046