Stable isotope probing analysis of the influence of liming on root exudate utilization by soil microorganisms

Abstract

Rhizosphere microorganisms play an important role in soil carbon flow, through turnover of root exudates, but there is little information on which organisms are actively involved or on the influence of environmental conditions on active communities. In this study, a 13CO2 pulse labelling field experiment was performed in an upland grassland soil, followed by RNA-stable isotope probing (SIP) analysis, to determine the effect of liming on the structure of the rhizosphere microbial community metabolizing root exudates. The lower limit of detection for SIP was determined in soil samples inoculated with a range of concentrations of 13C-labelled Pseudomonas fluorescens and was found to lie between 10(5) and 10(6) cells per gram of soil. The technique was capable of detecting microbial communities actively assimilating root exudates derived from recent photo-assimilate in the field. Denaturing gradient gel electrophoresis (DGGE) profiles of bacteria, archaea and fungi derived from fractions obtained from caesium trifluoroacetate (CsTFA) density gradient ultracentrifugation indicated that active communities in limed soils were more complex than those in unlimed soils and were more active in utilization of recently exuded 13C compounds. In limed soils, the majority of the community detected by standard RNA-DGGE analysis appeared to be utilizing root exudates. In unlimed soils, DGGE profiles from 12C and 13C RNA fractions differed, suggesting that a proportion of the active community was utilizing other sources of organic carbon. These differences may reflect differences in the amount of root exudation under the different conditions.

Exogenous glucosinolate produced by Arabidopsis thaliana has an impact on microbes in the rhizosphere and plant roots

“…For each gradient, one fraction representative of 13 C-labelled ('heavy') DNA and one fraction representative of unlabelled ('light') DNA were chosen according to a standardized method based on previous studies in the laboratory (Haichar et al, 2007(Haichar et al, , 2008 following buoyant density and DNA content of each fraction in comparison with control gradient containing unlabelled soil DNA and 13 C labelled DNA from Escherichia coli. The heavy fraction was considered to contain DNA representative of populations involved directly or not, in root exudates assimilation and the light one, to contain DNA from populations degrading soil organic matter or inactive (Figure 1) (Rangel-Castro et al, 2005;Haichar et al, 2008).…”

“…This culture-independent approach allows discriminating root exudate-assimilating microbial populations in plant rhizosphere from those dormant or degrading soil organic matter (Rangel-Castro et al, 2005;Haichar et al, 2008) and to link the identity of microorganisms to their function in soil (Radajewski et al, 2000;Lu et al, 2006).…”

Plant host habitat and root exudates shape soil bacterial community structure

“…The recent development of stable isotope probing (SIP) (Radajewski et al, 2000) and its application to tracking plant-derived C into microbial nucleic acids (Rangel-Castro et al, 2005;Lu et al, 2006;Prosser et al, 2006;Neufeld et al, 2007) or other biochemical markers (Treonis et al, 2004;Paterson et al, 2007) provide the opportunity to understand the functional diversity of plant-associated bacterial communities.…”

Who eats what, where and when? Isotope-labelling experiments are coming of age

“…In a recent study of soil cellulolytic bacteria, the 13 Clabelled cellulose was purified from the growth medium of Acetobacter xylinus, which was grown on 13 C-glucose as a sole source of carbon (el Zahar Haichar et al, 2007). Finally, 13 CO 2 -labelling of plants represents a clever approach to tracking plant-derived carbon to microorganisms in the rhizosphere (Cadisch et al, 2005;Lu and Conrad, 2005;Rangel-Castro et al, 2005). Such creative chemical and biological synthesis of complex organic compounds widens the application and versatility of SIP.…”

“…Regardless of this, what has been a common criticism, that is, 'cross-feeding' can now be turned into an advantage to look at the flow of carbon through ecosystems. For several SIP experiments, studying trophic networks was performed by labelling macroorganisms that provide nutrients for a natural microbial community (Cadisch et al, 2005;Lu and Conrad, 2005;Rangel-Castro et al, 2005) or by labelling microbial cells. Lueders et al (2006) recently conducted a cross-feeding study by supplementing indigenous soil microbial communities with an excess of 13 C-labelled E. coli cells.…”