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Materials and Methods
Plants, microcosms and incubation procedure
Three plants of different functional groups that are common and widely distributed in Europe, were selected as model plants (H. lanatus, L. cornicularius and P. lanceolata). Additionally, Z. mays (DEA, Pioneer France) was selected as an important culture plant in Europe and a model plant for rhizosphere research.
After surface sterilization (Benizri et al. 1995), seeds were transferred aseptically in Neff’s Modified Amoeba Saline (NMAS) (Page 1976) mixed 1:9 (volume:volume) with nutrient broth (NB) (Merck) (NB-NMAS) (100 µl) in 96’ well micro-titer-plates (Grainer, Germany) (except of Z. mays which was directly transferred on NB-NMAS Agar). Here seeds were allowed to germinate in the dark at 20°C. After germination, seedlings were transferred on 10% NB-NMAS Agar in Petri dishes and cultured at 21°C before aseptical transfer into the microcosms.
Figure 4. Microcosm set up: model plants (Holcus lanatus, Zea mays, Lotus corniculalus and Plantago lancelata) were grown in soil inoculated with protozoa free microbial rhizosphere microorganisms. The Amoeba treatment received axenic Acanthamoeba castellanii as microfaunal grazer. Soil was protected from airborne cysts of protozoa by wrapping sterile cotton wool around the basis of the shoot. To follow nitrogen uptake from soil into model plants 15N labelled Lolium perenne litter was added to the soil.
Preparation of microcosms and soil
Soil was collected from the upper 20 cm of a grassland site grown on a former agricultural field, which had been abandoned for more than 10 years (Van der Putten et al. 2000). The soil was taken in autumn and stored at 4 °C before sieving (4 mm) and use in the experiment. It contained 21.3 g kg-1 organic carbon, 1.27 g kg-1 total N, 0.33 g kg-1 total P and had a pH of 6.3.
15N labelled Lolium perenne leaf litter was produced as described by Wurst (2004). Before autoclaving, 15N labelled L. perenne leaf litter (C-to-N ratio 8.2) was homogeneously mixed with non labelled L. perenne litter (C-to-N ratio 11.5) to achieve litter containing 10 atom% 15N. To 250 g dry weight soil 0.39 g of the litter was added and mixed homogeneously. Prior to transfer of the soil into the microcosms it was autoclaved three times (20 min each, 121°C). Microcosms consisted of 250 ml polypropylen pots with a circular opening for plant shoots in the lid. Openings were sealed with sterile cotton wool to avoid contamination by airborne cysts of protozoa. A second opening was installed to improve aeration of the system (Figure 4).
Inoculation with bacteria and protozoa
A natural protozoa-free soil bacterial inoculum2 was prepared from the upper 20 cm soil from a grassland site grown on a former agricultural field, which had been abandoned for more than 10 years (van der Putten et al. 2000). The supernatant of a soil slurry (50 g fresh weight soil mixed 1:1 with NMAS on a horizontal shaker at 70 rpm for 20 min) was passed consecutively through two filters of 3 µm and then 1.2 µm (Bonkowski and Brandt 2002). The protozoa-free filtrate was cultured in NB-NMAS and checked for contamination for seven days by microscopic observation. Additionally one third of the filtrate was cultured in autoclaved soil. Both cultures were stored at 21°C in a climate chamber.
Prior adding 2 ml of protozoa-free filtrate to the microcosms, 5 g of the protozoa-free bacteria-soil culture was added into sterile soil of each microcosm. Subsequently, the soil was compressed to a density of 1.3 g cm-³ and incubated for 1 week at 24°C and 75 % relatively humidity in a climate chamber. Then, 0.78 g glucose was added to each pot in 3 ml aqueous solution to stimulate microbial activity. Prior to the addition of amoebae, small amounts of soil (covering the tip of a spatula) of each pot were mixed with NB-NMAS and checked for contamination for 7 days.
Axenic amoebae (Acanthamoeba castellanii) (Rosenberg 2008) were prepared following a modified protocol described by Bonkowski & Brandt (2002). Briefly, the amoeba culture was washed and centrifuged twice in NMAS (1000 rpm, 2.5 min). Protozoan treatments received 1 ml (approximately 8000 individuals) of the protozoa suspension, whereas the control treatments received 1 ml NMAS.
Plant transfer and cultivation
Seven days after protozoa inoculation, plants of similar size were selected and transferred into the microcosms under sterile conditions. Microcosms were then incubated in a climate chamber (18°C / 22°C night/ day temperature, 70% of humidity, 14 h of photoperiod, 460 ± 80 µmol m-2 s-1 photon flux density in the PAR range at plant level). Soil moisture was gravimetrically maintained at 70% of the water holding capacity by watering with sterile distilled water using a 0.02 µl syringe filter. Plant shoots were fixed in the opening of the microcosms with sterile cotton wool to avoid contamination with protozoa by air borne cysts.
Harvesting and analytical procedures
Plants were destructively harvested 21 days after transfer into the soil except for Z. mays which was harvested after 16 days to avoid root growth limiting conditions in the microcosms.
Plant leaf and root surface was scanned and analysed by WinFolia and WinRhizo software (Régent Instruments, Ottawa, Canada), respectively. Plant materials were subsequently freeze dried for biomass determination. Root adhering soil was taken as rhizosphere soil and separated from roots by handpicking. Subsamples of adhering soil were dried for water content determination (80°C, 48 h). Mineral N content was determined from 6 g root free adhering soil subsamples by extracting with 50 ml 0.5 M K2SO4 for 1 h at 130 rpm min-1 and subsequent filtering. Extracted samples were kept frozen until analysis. Mineral N (Nmin = NO3-N + NH4+-N) content of the K2SO4 extracts and measured in a Traax 2000 analyser (Bran and Luebbe).
Plant tissue and soil samples were milled to fine powder for analysis of total plant C and N as well as 15N/14N ratio by an elemental analyser (Carlo Erba, Na 1500 type II, Milan, Italy) coupled with an isotope mass spectrometer (Finnigan Delta S, Bremen, Germany). Data were presented as excess 15N compared to the natural abundance.
Total numbers of protozoa were enumerated by the most probable number technique (Darbyshire et al. 1974). Briefly, 5 g of soil were dispersed in 20 ml NMAS and shaken for 20 min at 75 rpm. Aliquots of 0.1 ml were added to microtiter plates and diluted two fold in 50 µl sterile NB-NMAS. Microtiter plates were incubated at 15°C and counted every second day for 21 days until protozoan numbers remained constant. Numbers were calculated according to Hurley and Roscoe (1983).
II.2.6. Statistical analysis
The effect of protozoa on the mobilization of N, plant N uptake and morphology of roots and leaves was analysed separately for each plant species with Amoeba as factor in SAS (v. 9.1) (n=7 for Zea mays and n=4 for H. lanatus and P. lanceolata, n=5 for bare soil). Normal distribution and homogeneity of variance were improved by log-transformation (Sokal and Rohlf 1995).
Table of contents :
CHAPTER I. INTRODUCTION
I.1. REGULATION OF CARBON PARTITIONING IN THE PLANT AND RHIZODEPOSITION
I.2. RHIZODEPOSITS: SOURCE OF ENERGY AND INFORMATION FOR MICROORGANISMS
I.3. PHOTOSYNTHATES ALLOCATION TOWARDS ROOT INFECTING AND FREE LIVING SYMBIONTS: AM FUNGI AND PROTOZOA
I.4. PROTOZOA – ARBUSCULAR MYCORRHIZAL FUNGI INTERACTIONS
I.5. OBJECTIVES
CHAPTER II. THE IMPACT OF PROTOZOA ON PLANT NITROGEN UPTAKE AND MORPHOLOGY VARIES WITH PLANT SPECIES
II.1. INTRODUCTION
II.2. MATERIALS AND METHODS
II.2.1. Plants, microcosms and incubation procedure
II.2.2. Preparation of microcosms and soil
II.2.3. Inoculation with bacteria and protozoa
II.2.4. Plant transfer and cultivation
II.2.5. Harvesting and analytical procedures
II.2.6. Statistical analysis
II.3. RESULTS
II.3.1. Plant growth as affected by Acanthamoeba castellanii
II.4. DISCUSSION
II.4.1. Conclusions
ACKNOWLEDEGMENTS
REFERENCES
CHAPTER III. EFFECTS OF PROTOZOA ON PLANT NUTRITION AND CARBON ALLOCATION DEPENDS ON THE QUALITY OF LITTER RESOURCES IN SOIL
III.1. INTRODUCTION
III.2. MATERIAL AND METHODS
III.2.1. Microcosms
III.2.2. Plants and incubation conditions
III.2.3. Preparation of 15N labelled plant litter
III.2.4. 13CO2 pulse labelling of plants
III.2.5. Plant and soil analyses
III.2.6. Analysis of the 13C/12C and 14N/15N ratios of soil and plant samples
III.2.7. PLFA patterns and lipid stable isotope probing
III.2.8. Counting of amoebae
III.2.9. Quantification of microbial N
III.2.10. Statistical analyses
III.3. RESULTS
III.3.1. Plant biomass, total C and N
III.3.2. Plant and microbial 15N and 13C enrichment
III.3.3. 13C enrichment of plant organs
III.3.4. 13C enrichment of belowground respiration
III.3.5. Phospholipid fatty acids
0Table of contents
III.3.6. δ13C signatures of PLFAs
III.4. DISCUSSION
III.4.1. Conclusions
ACKNOWLEDGEMENTS
REFERENCES
CHAPTER IV. PROTOZOA (ACANTHAMOEBA CASTELLANII) AND ARBUSCULAR MYCORRHIZAL FUNGI (GLOMUS INTRARADICES) MEDIATE THE PARTITIONING OF CARBON AND THE AVAILABILITY OF NITROGEN FOR PLANTAGO LANCEOLATA
IV.1. INTRODUCTION
IV.2. MATERIAL AND METHODS
IV.2.1. Microcosms, soil and microorganisms
IV.2.2. Plant preparation and growth conditions
IV.2.3. 13CO2 pulse labelling and quantification of 13C respiration of the belowground compartment
IV.2.4. Plant harvest and soil sampling
IV.2.5. Total C, N and isotope (13C/12C and 14N/15N) analyses of soil and plant samples
IV.2.6. Soil soluble mineral N (Nmin) concentration and microbial biomass
IV.2.7. Size and activity of the soil microbial community
IV.2.8. Microbial community structure
IV.2.9. Statistical analyses
IV.3. RESULTS
IV.3.1. Plant biomass and shoot-to-root ratio
IV.3.2. Total N, atom% 15N and total 15N in Plantago lanceolata
IV.3.3. Belowground respiration
IV.3.4. Soluble Nmin soil
IV.3.5. Microbial biomass, activity and community structure
IV.4. DISCUSSION
IV.4.1. Conclusions
ACKNOWLEDGEMENTS
REFERENCES
CHAPTER V. PROTOZOA AND ARBUSCULAR MYCORRHIZA COMPLEMENT EACH OTHER IN PLANT NITROGEN NUTRITION FROM A NUTRIENT PATCH
V.1. INTRODUCTION
V.2. MATERIAL AND METHODS
V.2.1. Microcosms and labelling procedure
V.2.2. Analytical procedures
V.2.3. Statistical Analysis
V.2.4. Results and Discussion
ACKNOWLEDGEMENTS
REFERENCES
CHAPTER VI. GENERAL DISCUSSION
