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Photosynthates allocation towards root infecting and free living symbionts: AM fungi and protozoa
From a plant perspective a tight control over its C budget should exist since lost C does not contribute to dry matter production. Contrary, from the microbial perspective, mechanisms to increase the net efflux of C from roots are likely to occur to maintain growth and activity (Bonkowski 2004). Even though plants transfer large amounts of photosynthates into the rhizosphere, competition for plant C between different root symbionts has been suggested (Vierheilig et al. 2000, Phillips et al. 2003). Competing symbionts can be separated into root infecting and free living microorganisms. We chose arbuscular mycorrizal (AM) fungi as a root infecting symbionts (Phylum Glomeromycota) (Schüßler et al. 2001). AM fungi form symbiosis with about 80% of all terrestrial plant genera (Smith and Read 1997) and about 60% of all land plants end up into symbiosis with arbuscular mycorrhizal fungi (Trappe 1987). Moreover AM fungi are suggested to be the oldest and most important terrestrial plant mutualists (Brundrett 2002). All mycorrhiza types possess two common features: an interface between plant root and fungal cells and extraradical hyphae extending into the soil (Johnson and Gehring 2007). Dead and living biomass of extraradical parts of AM fungi (e.g. spores and hyphae) make up 5 to 50 % of microbial biomass in agricultural soils (Olsson et al. 1999). AM fungi contribute predominantly to host plant phosphorous acquisition by increasing the absorptive area of roots via extraradical hyphae (Smith and Read 1997). In exchange for providing plants with mineral nutrients, the obligate biotroph AM fungi receive up to 30% of recently fixed plant assimilates (Johnson et al. 2002, Nguyen 2003, Jones et al. 2004, Heinemeyer et al. 2006). Thus, the mycelial system provides vital conduits for the translocation of nutrients from soil to plants and for reciprocal transfer of C from plant roots into extraradical AM hyphae (Smith and Read 1997). Recent studies demonstrate that AM fungi contribute to plant N gain by colonising litter patches (Hodge et al. 2000b, Hodge et al. 2001). However, there is no evidence that AM fungi have significant saprotrophic ability (Smith and Read 1997) and it is still unclear to what extend N allocation by AM fungi improves host plant performance (Johnson et al. 1997). The direct and rapid acquisition of photosynthetically fixed C and the rapid turnover of hyphal networks in soil (Staddon et al. 2003) suggest that mycorrhizal fungi form significant agents sequestering C in soil (Staddon 2005). The association between plant and AM fungi is mediated by the availability of nutrients, e.g. depends on the availability of soil N (Johnson and Gehring 2007). Moreover, plant growth responses on AM fungi association ranges in a continuum from positive (mutualism) to neutral (commensalism) and even to be negative (parasitism) (Johnson et al. 1997), but it is assumed that mutualism dominates (Marschner 1995, Smith and Read 1997). Species identity of the fungal partner determines plant nutrient supply that can directly affect plant growth (van der Heijden et al. 2003) and competitiveness of coexisting plant species. Additionally, terrestrial ecosystems contain many AM fungi and plant species that coexist in communities (Johnson et al. 1991, Allen et al. 1995, Sanders et al. 1996, Helgasson et al. 1998, Picone 2000, Ergeton-Warburton and Allen 2000). This indicates that the influence of AM fungi on plant growth is complex and, as shown recently, plant species and genotypes may vary in their responses to mycorrhizal colonization (Rillig et al. 2008). In conclusion, AM fungi contribute to complex belowground interactions and their activity influence the functioning and activity of other soil organisms that feed back to plant performance.
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).
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).
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).
The model microcosm system successfully protected protozoa contaminations from airborne cysts; no protozoan contaminations were found in the treatments. Due to poor establishment after transplanting, probably caused by the switch from axenic to microcosm conditions, only 4 of the initially 8 replicates per treatment could be used for P. lanceolata and H. lanatus and 7 for Z. mays. Lotus corniculatus did not establish after transfer of young seedlings into the microcosms. This might have been due to low mineral N in the soil and insufficient N2 fixing symbiotic bacteria (Rhizobia spp.) in the re-established microbial community (Lum and Hirsch 2003, Wurst and van Beersum 2008).
Plant growth as affected by Acanthamoeba castellanii
Protozoa did not significantly affect leaf and root biomass in the tested plant species (Table 2, Table 3). Specific root area of P. lanceolata and Z. mays increased in presence of amoebae by factors of 2.1 and 1.7, respectively (Figure 5). Additionally, the specific leaf surface of P. lanceolata increased 1.3-fold in presence of amoebae (Table 3, Figure 5). Generally, specific root area was lower for Z. mays than in H. lanatus and P. lanceolata indicating bigger and more compact roots of Z. mays. Holcus lanatus had the finest root system of tested plant species.
Plant species did affect the total numbers of A. castellanii in soil but the number peaked in planted soils where numbers were increased from 1089 ± 920 to 20185 ± 9184, respectively at the end of the experiment (F = 25.8, p < 0.0001). Mineral N concentration in rhizosphere soil was highly increased 2.7-fold in the presence of protozoa in bare soil but remained unaffected in planted soils (Table 2, Table 3).
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
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
III.3.6. δ13C signatures of PLFAs
III.4. DISCUSSION
III.4.1. Conclusions
ACKNOWLEDGEMENTS
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
CHAPTER VI. GENERAL DISCUSSION
REFERENCES
