Title: Survival of Rhizobium spp. in zink contaminated soils. Role of biotic and abiotic processes
Other Titles: Overleving van Rhizobium spp. in zink verontreinigde bodems. Rol van biotische en abiotische processen
Authors: Boonen, Miet; M9919229
Issue Date: 19-Dec-2012
Abstract: About 40% of the European production of sewage sludge, the solid waste product of wastewater treatment, is applied as a fertilizer to agricultural land. Sewage sludge contains toxic trace metals such as zinc (Zn) and copper (Cu). Long-term application of sewage sludge can enrich soils with these metals to concentrations that can be harmful for the soil ecosystem. The agronomically important nitrogen fixation process by legumes such as white clover (Trifolium repens L.) appears very sensitive to Zn derived from sewage sludge. This is ascribed to the effects of Zn on survival of the microsymbiont Rhizobium leguminosarum in soil. The aim of this study was to examine why Rhizobium leguminosarum spp. is particularly sensitive to Zn. Experiments were setup to examine biotic and abiotic factors affecting survival of Rhizobium leguminosarum spp. under Zn stress. The hypothesis was that the high sensitivity is mainly related to biotic interactions in the soil ecosystem and to the heterogeneities of Zn distribution in sludge amended soils. The population size of Rhizobium leguminosarum spp. in soil depends on predation by protozoa or competition with other soil micro-organisms that generally grow faster than Rhizobium leguminosarum spp.. Such interactions may exceed a tipping point under Zn stress, especially when the host plant is absent. The study included the development of a relatively fast and easy technique to enumerate Rhizobium leguminosarum spp. in soil.The plant infection method is commonly used to estimate the most probable number (MPN) of Rhizobium leguminosarum spp. in soils. This technique estimates viable numbers and is predictive for nitrogen fixation, however it is time-consuming and not selective enough to detect a specific Rhizobium leguminosarum bv. trifolii strain. More rapid qPCR assays have been developed for nitrogen fixing bacteria, however it is unknown to what extent this assay correlates with viable cell counts in survival experiments. Two qPCR assays were setup with newly developed primers. A first one to quantify specific Rhizobium leguminosarum bv. trifolii ANU843 and a second one for the quantification of all Rhizobium leguminosarum species. Numbers of Rhizobium leguminosarum in liquid cultures detected by the qPCR protocols were not different from plate count or plant infection counts. Detection limits of qPCR protocols in soil were 1.2×104 (ANU) and 4.2×103 (RHIZ) cells per g soil. A standard addition assay with the Rhizobium leguminosarum bv. trifolii ANU843 strain in 2 soils showed average recoveries of 230% using the ANU primer set. However, indigenous Rhizobium leguminosarum numbers in 15 grassland soils as quantified by qPCR correlated significantly (p<0.05) but weakly (R2= 0.30 in log-log plot) with MPN and qPCR overestimated MPN by, on average, a factor of 12. The overestimation of Rhizobium leguminosarum bv. trifolii numbers by qPCR could be due to the presence of viable but non-nodulating cells, deceased cells or extracellular DNA targeted with qPCR but not with MPN. A soil incubation experiment with viable cells, non-viable cells (autoclaved) and isolated DNA showed that DNA or non-viable cells were detected in soil up to 6 months after addition and incubation at 20&#176;C in moist conditions. The qPCR assay appears robust and accurate in freshly inoculated soils, however this study cast doubts about its accuracy for long-term rhizobial survival studies. Validation with soil-plant studies across a wide range of conditions is required before qPCR of rhizobia in soil can be advocated as an ecologically relevant assay for studies in the soil.Recent (2008) long-term field data in UK showed a larger effect of soil Zn on survival of Rhizobium leguminosarum bv. trifolii in sewage sludge amended soils compared to corresponding Zn salt amended soils and this is counterintuitive based on Zn bioavailability principles. In this study, we tested the hypothesis that sludge particles act as hotspots of Zn contamination and that these are more lethal to Rhizobium leguminosarum spp. than a homogeneously mixed Zn contamination. First, sludge was either mixed or localized as a layer in soil columns and either the soil or the localized sludge was spiked with Zn salt. Secondly, soil was either spiked with Zn salt or was contaminated with Zn spiked sludge that was localized in the middle of a soil layer. The cell numbers in the column experiment, either based on qPCR or on the plant infection method, declined during 10 months of incubation and were neither affected by sludge localization method nor by Zn, however these numbers were all smaller than in a control soil (without Zn contamination) unamended with sludge. In the disc experiment, qPCR based numbers in soil at 11 months after soil inoculation were significantly smaller near Zn contaminated sludge than near clean sludge or in Zn spiked soil not containing sludge at equivalent average Zn. However, that difference was small (<1 log unit) and was certainly smaller than the overall effects of time on survival. It is concluded that sludge has an overall adverse effect on Rhizobium leguminosarum spp. but that the soil heterogeneities have no markedly larger effect than homogeneous contaminations within 1 year. Longer term studies are required to test mechanisms that affected rhizobia in the field experiments.The sensitive effects of soil contamination with Zn on symbiotic nitrogen fixation by white clover are ascribed to its effects on the survival of Rhizobium leguminosarum bv. trifolii spp. in the soil, especially when the host plant has been absent for a long time. A protective effect of the host plant was suggested. The effect of the host plant on survival and genetic diversity of rhizobial communities in Zn contaminated soils was experimentally tested. Survival (qPCR) and community structure (DGGE) of rhizobia were monitored during 18 months in a pot trial with a factorial combination of soil metal contamination (60 – 1600 mg Zn kg-1) and plant species (white clover, ryegrass and a mixture of both) and this for a freshly Zn spiked soil (LOV) and a long-term trace metal contaminated soil (AL). Both soils were inoculated at the start. Survival (18 months) of Rhizobium leguminosarum spp. was larger and less sensitive to Zn in soils under clover than under grass, however the effects of soil Zn on abundance in grass planted soils were smaller than in long-term field studies reported before. The DGGE fingerprints of the rhizobial communities revealed no (LOV) to small (AL) effect of Zn in clover planted soils whereas significant gradients were found in grass-only planted soils. This study experimentally confirms the positive effect of clover on Rhizobium leguminosarum communities in Zn contaminated soils. Ecological experiments were setup to unravel if the effect of Zn on Rhizobium leguminosarum spp. is related to a larger sensitivity to Zn compared to other soil micro-organisms, using Pseudomonas putida as a model, or to an ecological disadvantage of Rhizobium leguminosarum spp. in soils. In C limited liquid single cultures, Rhizobium leguminosarum bv. trifolii ANU843 was more sensitive to Zn than Pseudomonas putida KT2440 during net growth but remained culturable for a longer time during stationary phase. Tested in combination in liquid cultures, Pseudomonas putida KT2440 appeared to be less sensitive to Zn than Rhizobium leguminosarum bv. trifolii ANU843, which failed to maintain its population during the stationary phase at moderate Zn stress (0.5 and 10 mg Zn l-1), likely as a result of competition for available C with Pseudomonas putida KT2440. Survival (12 months) of these species inoculated in isolation in sterile soil confirmed larger sensitivity to Zn of Rhizobium leguminosarum spp. compared to Pseudomonas putida spp., however no such differences were found when both bacteria were inoculated in combination in non-sterile soils. Non-sterile soils amended with plant residue further reduced survival of both inoculants under Zn stress and this was most pronounced for Rhizobium leguminosarum spp. for which cell density decreased from 7.3 initially to 4.2 log numbers after 12 months. In clover planted soils, Rhizobium leguminosarum spp. survived markedly better than in unplanted soils up to moderate Zn stress in soil (500 mg Zn/kg), not affecting clover growth whereas the reverse was true for Pseudomonas putida spp.. It is concluded that Rhizobium leguminosarum bv. trifolii ANU843 is slightly more sensitive to Zn than Pseudomonas putida KT2440 in isolation but that ecological disadvantages likely explain their gradual extinction in the absence of the host plant under moderate Zn stress in the field. Summarizing, this study confirmed that the host plant (clover) has a protective effect on the survival of Rhizobium leguminosarum bv. trifolii in Zn contaminated soils. In the absence of the host plant, the survival of Rhizobium leguminosarum bv. trifolii becomes particularly sensitive to Zn contamination (abiotic stress) under increasing competition with other soil organisms (biotic stress). Longer-term studies are required to reveal the role of Zn hotspots in sludge amended soils on Rhizobium leguminosarum survival.
ISBN: 978-90-8826-274-6
Publication status: published
KU Leuven publication type: TH
Appears in Collections:Division Soil and Water Management
Centre of Microbial and Plant Genetics

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