Decomposer-plant interactions: Effects of Collembola on plant performance and competitiveness Vom Fachbereich Biologie der Technischen Universität Darmstadt zur Erlangung des akademischen Grades eines Doctor rerum naturalium genehmigte Dissertation von Dipl. Biol. Kerstin Endlweber aus Frankfurt a. M. Berichterstatter: Prof. Dr. Stefan Scheu Mitberichterstatter: Prof. Dr. Gerhard Thiel Tag der Einreichung: 29.05.2007 Tag der mündlichen Prüfung: 10.07.2007 Darmstadt 2007 D17 Never does nature say one thing and wisdom another. Juvenal LIST OF PULICATIONS LIST OF PUBLICATIONS Endlweber K, Scheu S (2006) Effects of Collembola on root properties of two competing ruderal plant species. Soil Biology & Biochemistry 38: 2025- 2031. (Chapter 2) Endlweber K, Scheu S (2007) Interactions between mycorrhizal fungi and Collembola: effect on root structure of competing plant species. Biology and Fertility of Soils 43: 741-749. (Chapter 3) Endlweber K, Ruess L, Scheu S (2007) Dietary routing in Collembola: Determining Collembola feeding preferences by stable isotope and compound specific fatty acid analysis. (in preparation) (Chapter 4) Endlweber K, Schäffner AR, Scheu S (2007) Analysis of decomposer-induced changes of gene expression profiles in Arabidopsis thaliana. (in preparation) (Chapter 5) OTHER PUBLICATIONS Endlweber K, Scheu S (2006) Establishing arbuscular mycorrhiza-free soil: A comparison of six methods and their effects on nutrient mobilization. Applied Soil Ecology 34: 276-279. Birkhofer K, Endlweber K, Gavish E, Lubin YD, von Berg K, Wise D and Scheu S (2007) Spider functional group diversity does not improve aphid suppression in a winter wheat field in central Germany. (in preparation) TABLE OF CONTENTS TABLE OF CONTENTS ZUSAMMENFASSUNG......................................................................................................1 ABSTRACT ....................................................................................................................3 CHAPTER 1 ...................................................................................................................5 GENERAL INTRODUCTION ...............................................................................................5 1.1 Terrestrial ecosystems................................................................................................................................... 5 1.2 Effects of decomposers on plants.................................................................................................................. 6 1.3 Plant performance and mycorrhiza................................................................................................................ 7 1.4 Plant nutrient uptake, plant competition and root morphology ..................................................................... 9 1.5 Collembola .................................................................................................................................................. 10 1.6 Objectives.................................................................................................................................................... 13 CHAPTER 2 .................................................................................................................14 EFFECTS OF COLLEMBOLA ON ROOT PROPERTIES OF TWO COMPETING RUDERAL PLANT SPECIES ......................................................................................................................14 2.1 Abstract ....................................................................................................................................................... 14 2.2 Introduction................................................................................................................................................. 15 2.3 Materials and Methods................................................................................................................................ 17 2.4 Results......................................................................................................................................................... 19 2.4.1 Collembola........................................................................................................................................... 19 2.4.2 Plant performance ................................................................................................................................ 19 2.5 Discussion ................................................................................................................................................... 23 2.6 Conclusions................................................................................................................................................. 26 CHAPTER 3 .................................................................................................................27 INTERACTIONS BETWEEN MYCORRHIZAL FUNGI AND COLLEMBOLA: EFFECTS ON ROOT STRUCTURE OF COMPETING PLANT SPECIES ...................................................................27 3.1 Abstract ....................................................................................................................................................... 27 3.2 Introduction................................................................................................................................................. 28 3.3 Materials and Methods................................................................................................................................ 29 3.4 Results......................................................................................................................................................... 32 3.4.1 Plant combination ................................................................................................................................ 32 3.4.2 Effects of mycorrhiza........................................................................................................................... 35 3.4.3 Effects of Collembola .......................................................................................................................... 37 3.5 Discussion ................................................................................................................................................... 38 3.5.1 Plant combination ................................................................................................................................ 38 3.5.2 Mycorrhiza ........................................................................................................................................... 39 3.5.3 Collembola........................................................................................................................................... 40 3.6 Conclusions................................................................................................................................................. 42 CHAPTER 4 .................................................................................................................43 DIETARY ROUTING IN COLLEMBOLA: DETERMINING COLLEMBOLA FEEDING PREFERENCES BY STABLE ISOTOPE AND COMPOUND SPECIFIC FATTY ACID ANALYSIS ..............................43 4.1 Abstract ....................................................................................................................................................... 43 4.2 Introduction................................................................................................................................................. 44 4.3 Materials and Methods................................................................................................................................ 46 4.5 Results......................................................................................................................................................... 49 4.5.1 Stable Isotopes ..................................................................................................................................... 49 4.5.1.1 Plants................................................................................................................................................. 49 4.5.1.2 Collembola........................................................................................................................................ 49 4.5.2 Fatty acids ............................................................................................................................................ 51 4.6 Discussion ................................................................................................................................................... 54 CHAPTER 5 .................................................................................................................58 DECOMPOSER ANIMALS (COLLEMBOLA) INDUCE THE EXPRESSION OF DEFENCE AND AUXIN GENES IN PLANTS.........................................................................................................58 5.1 Abstract ....................................................................................................................................................... 58 5.2 Introduction................................................................................................................................................. 59 5.3 Materials and Methods................................................................................................................................ 60 5.3.1 Plant growth experiment ...................................................................................................................... 61 5.3.2 Microarray experiment......................................................................................................................... 61 5.3.3 DNA array hybridisation...................................................................................................................... 63 5.3.4 Statistical Analysis............................................................................................................................... 64 5.4 Results......................................................................................................................................................... 64 5.4.1 Plant growth ......................................................................................................................................... 64 5.4.2 Gene expression ................................................................................................................................... 65 5.5 Discussion ................................................................................................................................................... 69 5.6 Conclusions................................................................................................................................................. 73 GENERAL DISCUSSION..................................................................................................74 REFERENCES ..............................................................................................................81 ACKNOWLEDGEMENTS .................................................................................................98 CURRICULUM VITAE......................................................................................................99 ZUSAMMENFASSUNG 1 ZUSAMMENFASSUNG In Laborexperimenten unterschiedlicher Komplexität wurde der Einfluss von Collembolen auf Pflanzenwachstum und Konkurrenz zwischen Pflanzen untersucht. Das Konkurrenzverhältnis zwischen Pflanzenarten wird durch Unterschiede in der Wurzelmorphologie und Nährstoffaufnahme bestimmt. In einem Experiment wurde überprüft, ob Collembolen das Konkurrenzverhältnis zwischen Cirsium arvense und Epilobium adnatum durch Veränderungen der Nährstoffverfügbarkeit verschieben. Collembolen beeinflussten weder die Biomasse noch die Nährstoffgehalte beider Pflanzenarten, induzierten jedoch die Bildung längerer, dünnerer Wurzeln und erhöhten die Anzahl an Wurzelspitzen, insbesondere bei E. adnatum. In einem zweiten Experiment wurde überprüft, ob Collembolen über den Fraß an Mykorrhizapilzen das Konkurrenzverhältnis zwischen Lolium perenne und Trifolium repens verändern. Collembolen reduzierten die Konkurrenzkraft von L. perenne gegenüber T. repens, diese Reduktion war jedoch unabhängig vom Mykorrhizierungsgrad der Pflanzenwurzeln. Wie im ersten Experiment induzierten Collembolen die Bildung von längeren, dünneren Wurzeln und erhöhten die Anzahl von Wurzelspitzen in beiden Pflanzenarten, wobei die Wirkung bei L. perenne stärker ausgeprägt war. Die Veränderungen in der Wurzelmorphologie durch Collembolen beruhten wahrscheinlich auf der Verletzung von Wurzeln beim Fraß der Tiere in der Rhizosphäre und auf der Schaffung nährstoffreicher Bereiche im Boden. Zur Klärung ob Collembolen direkt an Wurzeln fressen wurde in einem dritten Experiment der Kohlenstofffluss von Pflanzenwurzeln in Collembolen mit Hilfe stabiler Isotope und komponentenspezifischer Fettsäure-Analyse untersucht. Als Versuchspflanze wurde Mais (C4-Pflanze) verwendet, dem 15N markierte C3-Streu zugegeben wurde. Collembolen nahmen bevorzugt Kohlenstoff und Stickstoff aus ZUSAMMENFASSUNG 2 Pflanzen auf. Ihr Gehalt an Kohlenstoff und Stickstoff wurde durch die Verfügbarkeit von Pflanzen sowie durch Streu gesteigert, allerdings führte die Kombination beider Ressourcen nicht zu einer weiteren Steigerung. Collembolen nahmen bevorzugt Fettsäuren aus Pflanzenmaterial auf, ihr Fettsäuremuster wurde durch die Verfügbarkeit von Streu nicht beeinflusst. Durch Verletzung oder Fraß an Pflanzenwurzeln werden sowohl die Abwehr als auch die Bildung von Botenstoffen in Pflanzen induziert. Durch Verwendung von DNA- Micorarrays wurde die Induktion von Pflanzenabwehr und Produktion von Phytohormonen in Arabidopsis thaliana durch Collembolen überprüft und diese mit Veränderungen im Rosettenwachstum korreliert. Collembolen reduzierten anfänglich das Wachstum von A. thaliana, die Pflanzen kompensierten diese Wachstumsverzögerung jedoch während der weiteren Entwicklung. Die temporäre Wachstumsdepression beruhte vermutlich auf einer Induktion von Abwehrstoffen durch Collembolen. Die Wachstumsverzögerung wurde später jedoch vermutlich durch eine erhöhte Produktion von Auxin ausgeglichen. Die Ergebnisse der Arbeiten weisen daraufhin, dass Collembolen Pflanzen hauptsächlich über zwei Mechanismen beeinflussen: (i) Durch Beweidung von Mikroorganismen in der Rhizosphäre verändern sie die Verfügbarkeit sowie Verteilung von Nährstoffen im Boden und fördern so die Nährstoffversorgung und das Wachstum von Pflanzen; (ii) durch Weiden in der Rhizosphäre induzieren Collembolen die Abwehr von Pflanzen gegen Schädlinge und erhöhen so den Schutz vor Fraßfeinden. Die dadurch bedingte Wachstumsverzögerung wird durch die von Collembolen induzierte Bildung von Wachstumshormonen ausgeglichen. Das kompensatorische Wachstum wird durch höhere Nährstoffverfügbarkeit und ein expansiv wachsendes Wurzelsystem in Anwesenheit von Collembolen ermöglicht. ABSTRACT 3 ABSTRACT This study investigated the effect of Collembola on plant growth and competition between plant species. The competitive relationship between plants is determined by differences in root morphology and foraging strategy. In the first experiment the effect of Collembola on the competitive relationship between Cirsium arvense and Epilobium adnatum was analysed. Collembola neither affected plant biomass nor plant nutrient concentration. However, they induced the production of longer, thinner roots and enhanced the number of root tips with the effect being more pronounced in E. adnatum. The inoculation of plant roots with mycorrhizal fungi is an important factor improving plant nutrition. The second experiment focused on changes in the competitive relationship between Lolium perenne and Trifolium repens due to Collembola grazing on mycorrhizal fungi. Collembola reduced the competitive superiority of L. perenne over T. repens. However, the reduction was independent of the rate of mycorrhizal inoculation. Similar to the first experiment Collembola induced the production of longer, thinner roots and increased the number of root tips, particularly in L. perenne. The results suggest that Collembola affect root morphology by damaging plant roots while grazing in the rhizosphere and by creating nutrient rich patches in soil. In a third experiment feeding preferences of Collembola were investigated using analysis of stable isotopes and compound specific analysis of fatty acids. Collembola were introduced to a system consisting of a maize plant (C4 plant), growing in soil mixed with 15N labelled C3 litter. Collembola preferentially incorporated plant-born carbon and nitrogen. The concentration of C and N in Collembola tissue increased when either litter or plants were available, whereas the combination of both resources caused no further increase in nutrient concentrations. Collembola ABSTRACT 4 preferentially incorporated fatty acid originating from plant material, whereas the fatty acid composition of Collembola was not affected by the availability of litter. Damaging or feeding on plant roots induces plant defence as well as the production of plant hormones. In a fourth experiment induction of plant defence and phytohormone production in Arabidopsis thaliana by Collembola were investigated employing DNA microarrays. Gene expression patterns were correlated with changes in rosette growth. Collembola initially reduced growth of A. thaliana with the deceleration being compensated during further development. The temporary deceleration probably was caused by the induction of plant defence by Collembola thereby reducing plant investment in growth. The enhanced production of the growth promoting hormone auxin presumably facilitated the compensational growth during further development. The results of this study suggest that Collembola influence plant growth mainly via two mechanisms: (i) Collembola affect nutrient availability and distribution by grazing on microorganisms in rhizosphere and thus enhance plant nutrition and growth; (ii) while grazing in the rhizosphere, Collembola induce plant secondary metabolism and thus increase plant defence against herbivores. The subsequent deceleration in plant growth is compensated by the increased production of growth promoting hormones in presence of Collembola. Further, the compensational growth is facilitated by the increased nutrient availability and the production of a more expansive root system induced by Collembola. CHAPTER 1 5 CHAPTER 1 GENERAL INTRODUCTION 1.1 Terrestrial ecosystems Terrestrial ecosystems consist of two compartments, the above- and below-ground system. Both subsystems are intimately linked by plants, forming the nutritional basis for above- and below-ground biota. Plant growth depends on decomposer communities, which break down organic matter and mobilise nutrients for plant uptake. By increasing the availability of nutrients soil organisms therefore promote plant growth and performance, and subsequently affect their own resource (Wardle 2002). Soil organisms are assumed to be food limited (Hairston 1989) and thus strong competition and niche differentiation should structure below-ground systems. However, there is limited experimental evidence that competition is the major force structuring soil communities (Scheu and Setälä 2002). Even though many decomposers use similar resources, species tend to switch between alternative food sources. Therefore, below-ground systems are characterised by a low degree of specialisation with most soil organisms being general feeders on a broad spectrum of food sources (Maraun et al. 2003). This low proportion of specialist feeders is due to the absence of coevolution between soil organisms and dead organic matter, the basic resource in soil (Scheu and Setälä 2002). As a consequence ascribing soil organisms to distinct trophic levels in food webs is an “insufficient” approach for structuring soil systems. Alternatively soil organisms are often classified according to body size. Body size restricts the mobility in soil and also determines what resources can be exploited. In this classification system bacteria and fungi (microflora) form the CHAPTER 1 6 smallest size class followed by microfauna comprising e.g. nematodes and protozoa (body size less than 0.1 mm). The mesofauna consist of e.g. Collembola, mites and other soil animals with a body width between 0.1 mm and 2 mm. Earthworms, diplopods and chilopods are classified as macrofauna (body width greater than 2 mm). 1.2 Effects of decomposers on plants Plants and decomposers are linked by a complex system of direct and indirect interactions. Direct effects include antagonistic interactions, such as root feeding and pathogen infection, but also mutualistic interrelationships between plant and root biota such as mycorrhiza. Indirect interactions affect plant performance through several mechanisms, such as modification of physical properties of soil and seed viability. However, the predominant indirect interaction includes the mineralisation and thus the mobilisation of nutrients for plant uptake (Bremer and Vankessel 1992). The microflora (fungi and bacteria) is of fundamental importance for mineralisation processes. Microorganisms break down organic matter, mineralise complex compounds to simpler forms and immobilise the mineralised nutrients by incorporation into microbial biomass (Jonasson et al. 1996). Plants stimulate the mineralisation by allocating up to 20% of the photosynthetically fixed carbon as root exudates to the rhizosphere (Nguyen 2003). The high availability of carbon compounds triggers the increased density and activity of the mainly carbon limited soil organisms in the rhizosphere compared to the bulk soil (Wardle 1992). The increased activity of micro-decomposers and their respective grazers promotes nutrient mineralisation and ultimately increases plant nutrient acquisition (Clarholm 1985). CHAPTER 1 7 Depending on soil fertility either a fungal- or a bacterial-based energy channel is facilitated (Wardle et al. 2004). Bacteria based systems in fertile soils support rapid nutrient cycling due to high turnover rates, whereas in fungal based systems the turnover rate is slow and nutrients are immobilised in fungal biomass (Wardle et al. 2004). Microflora activity during such processes is influenced by higher level decomposers e.g. microfauna, meso- and macrofauna (Beare et al. 1992). Microbial grazers affect microbial turnover but also influence competition and composition of the microbial community (Tiunov and Scheu 2005). The soil fauna might shift the fungal to bacterial ratio by influencing the competitive relationship between fungi and bacteria (Hanlon and Anderson 1979) an interaction that consequently affects the rate of nutrient mineralisation (Scheu et al. 1999, Bonkowski et al. 2001). Decomposers also modify the spatial distribution of nutrients in soil, as earthworms mix soil and organic matter (Wurst et al. 2003, Kreuzer et al. 2004). Soil organisms may also create nutrient rich patches by casting (Sjursen and Holmstrup 2004). Particularly earthworm middens are zones of high microbial activity and form microhabitats for other soil organisms (Maraun et al. 1999, Tiunov and Scheu 2000). The mineralisation of nutrients und thus the nutrient availability for plants depends on a complex interplay of indirect interactions that structure the soil food web. This system of close interrelationships between plants and soil organisms has not been sufficiently investigated and therefore its functioning is little understood. 1.3 Plant performance and mycorrhiza Arbuscular mycorrhizal (AM) fungi form symbiosis with about 80% of all terrestrial plant genera (Smith and Read 1997). Their widespread hyphal network extends the absorptive surface of the root system and facilitates plant nutrient acquisition. The symbiosis is associated with the allocation of up to 20% of the photosynthetically CHAPTER 1 8 fixed carbon to the root symbiont (Wang et al. 1989). Indeed, photosynthetically inactive plants are not inoculated with mycorrhizal fungi (Lerat et al. 2002). The AM fungus enables the plant to take up relative immobile ions such as phosphorus but also improves water uptake. Recent studies demonstrated a translocation of nitrogen from the AM fungus to the plant root. Mycorrhizal fungi may directly gain nutrients from organic matter and allocate these to the plant (Perez-Moreno and Read 2000). Therefore, plants do not exclusively depend on nitrogen mineralisation by free living soil microorganisms. Nevertheless, the availability of nitrogen is a major factor limiting plant growth and it is still unclear to what extend nitrogen allocation by AM fungi improves plant performance (Johansen 1999). The symbiosis between plants and AM fungi depends on the availability of nutrients; a high nutrient availability might reduce the effectiveness of the symbiosis (Klironomos et al. 1996). Under such conditions the mycorrhizal association may turn from mutualism into parasitism (Johnson et al. 1997). Mycorrhizal fungi differ in their ability to supply plants with nutrients with strong consequences for plant growth (Smith et al. 2000, van der Heijden et al. 2003). Their performance optimum is determined by abiotic factors, such as pH and soil moisture content as well as nutrient availability (van Aarle 2002). Species identity of the fungal partner mainly determines plant nutrient acquisition and subsequently plant growth and performance. However, plants also vary in their dependency on nutrients supplied by mycorrhizal symbiosis. The effectiveness of nutrient acquisition determines plant growth and performance and therefore the competitiveness. Thus, plant coexistence as well as competitive superiority might be influenced by mycorrhizal inoculation (van der Heijden et al. 2003). CHAPTER 1 9 As soil organisms AM fungi are part of the complex system of interactions below ground, the activity of other soil biota might influence the functioning of mycorrhizal association and consequently affect plant performance. Soil invertebrates could reduce the effectiveness of mycorrhiza by grazing on hyphae and reducing hyphal density (Setälä 1995). Faunal activity might also improve nutrient availability for plants by stimulation of root colonisation and by dispersing mycorrhizal spores (Gange 1993, Klironomos and Kendrick 1996). 1.4 Plant nutrient uptake, plant competition and root morphology Plant nutrient uptake is mainly governed by the morphology of the root system. The heterogeneous distribution of nutrients in soil forces plant roots to proliferate into nutrient rich patches. Efficient root foraging is probably a trade off between developing and sustaining an extensive root system and precise proliferation into nutrient rich patches (Campbell et al. 1991). The root foraging potential of plants differs between species and therefore has important implications for plant competition (Hutchings et al. 2003). Fast growing plants with an extensive root system might reach and exploit nutrient rich patches quickly and suppress smaller plants in heterogeneous soils (Rajaniemi and Reynolds 2004). Plant root structure and morphology is affected by competition with other plant roots (Maina et al. 2002). Plants increase root production in presence of other roots to gain a competitive advantage in exploiting resources but preferentially proliferate roots in unoccupied areas to avoid root competition (Gersani et al. 2001). Root morphology and structure can be affected by soil organisms such as root microorganisms or soil invertebrates. The activity of soil invertebrates might affect nutrient availability resulting in an increased root growth (Canellas et al. 2002). The facilitation of nutrient uptake by mycorrhizal fungi enables the plant to reduce the root CHAPTER 1 10 system (Wulf et al. 2003). Bonkowski et al. (2001) demonstrated that plants reduce the length of roots as well as the number of root tips when inoculated with mycorrhizal fungi. 1.5 Collembola Collembola are an ubiquitous arthropod group with 7500 described species worldwide. They colonise a large range of terrestrial ecosystems from arid habitats such as the Antarctica and sand deserts to the humid zones of sea coasts and rivers (Usher and Booth 1986; Andre et al. 1997). The characteristic morphological feature of Collembola is the furca, which enables the animals to cover distances that span many times their own body length. The furca is an effective mechanism that allows Collembola to escape predators. However, several mainly edaphic Collembola species have reduced or lost the furca. Collembola belong to the mesofauna and are generally of small size. Euedaphic species grow up to 10 mm but most edaphic species reach only a few millimetres. In terrestrial habitats they are particularly abundant in soil and in the litter layer. In soil Collembola reach densities ranging between 50.000/m² in agricultural systems and up to 1 Mio /m² in boreal coniferous forests (Peterson and Luxton 1982). Collembola are mainly regarded as saprophageous or microphytophageous, but more likely are food generalists (Hopkin 1997). They feed on decaying plant material, bacteria, algae, pollen and also on living plant material (Wolters 1985, Chen et al. 1995). A few species feed on nematodes or eggs of other Collembola (Lee and Widden 1996). Analysis of stable isotope signatures indicates that Collembola inhabit a wide range of trophical niches (Chahartaghi et al. 2005). However, feeding habits are influenced by the availability of food resources and therefore depend on both, habitat and season (Anderson and Healey 1972, Wolters 1998). CHAPTER 1 11 Edaphic Collembola are particularly active in the rhizosphere of plants. Albers et al. (2006) demonstrated that plant born carbon is rapidly incorporated into Collembola, indicating that their food resources are either closely related to root exudates or to microorganisms utilising root derived carbon, e.g. mycorrhizal fungi. Collembola probably graze on hyphae of mycorrhizal fungi (Moore et al. 1987). The effect of Collembola on the mutualistic relationship between plant and fungus has been demonstrated to be either positive or negative depending on Collembola density (Bakonyi et al. 2002). However, by affecting the mycorrhizal fungus inoculation they might influence plant nutrition and ultimately affect plant growth and performance (Kaiser and Lussenhop 1991, Gange 2000). Changes in plant nutrition and performance might also be due to indirect effects. By grazing on microorganisms in the rhizosphere of plants Collembola affect nutrient cycling by changing the microbial activity and community composition (Bardgett et al. 1993, Chen et al. 1995, Maire et al. 1999, Petersen 2002). They might also increase nutrient availability by excretion and faecal pellet deposition (Petersen 2000, Sjursen and Holmstrup 2004). As a consequence, Collembola also affect nutrient availability to plants and therefore plant growth and nutrient content (Lussenhop 1992, 1996, Filser 2002). Effects of Collembola on plant nutrient content cascade up and ultimately affect above-ground phytophagous insects (Scheu et al. 1999, Wurst and Jones 2003). The influence of Collembola on plant nutrition probably varies with plant species and functional group. Plant nutrient acquisition is closely related to the root morphology and inoculation with root biota. Resource availability in soil varies with space and time. The reaction of plants to heterogeneous resource distribution depends on their functional group and root morphology. Many plant species increase their nutrient uptake by proliferating roots in nutrient rich patches (Hodge et al. 1998, Hodge 2004). Thus nutrient acquisition depends on plant species and influences the CHAPTER 1 12 competition between plant species (Hodge 1999). The patchiness of nutrients as well as the overall availability is probably influenced by Collembola. Hence, Collembola might affect nutrition of plant species or functional groups differently and thus affect plant competition. CHAPTER 1 13 1.6 Objectives This study investigated the effect of Collembola on plant performance and competitiveness. In a first experiment the impact of Collembola on plant performance and competition was determined by comparing morphological and growth related parameters e.g. plant biomass, nutrient content and root morphology (Chapter 2). The experiment tested the hypothesis that Collembola affect the competitive ability of different plant species by altering nutrient availability and individual plant performance. The results of the first experiment were followed up in a second approach focusing on plant nutrient acquisition as influenced by mycorrhizal fungi (Chapter 3). Analysing nutrient contents, root morphology and the inoculation of plant roots with mycorrhizal fungi of Trifolium repens and Lolium perenne, the hypothesis was tested that Collembola affect competition between plants of different functional groups by influencing the interaction between plants and mycorrhizal fungi. In a third approach Collembola feeding habits were determined by a combined analysis of Collembola stable isotope signatures of nitrogen (15N/14N) and carbon (13C/12C) as well as fatty acid composition and compound specific analysis (Chapter 5). Collembola were introduced in pots with Maize plants growing in soil mixed with labelled (15N) litter of Lolium perenne L (C3 plant). Fatty acid and stable isotope analysis provided information about the resources used by Collembola and connected this data with information derived from earlier experiments. The fourth experiment related the morphological response of Arabidopsis thaliana to changes in gene expression. Using a custom made DNA-Microarray the effect of Collembola on plant secondary metabolism and hormone production was investigated (Chapter 4). CHAPTER 2 14 CHAPTER 2 EFFECTS OF COLLEMBOLA ON ROOT PROPERTIES OF TWO COMPETING RUDERAL PLANT SPECIES 2.1 Abstract Plant roots compete for nutrients mineralised by the decomposer community in soil. By affecting microbial biomass and activity Collembola influence the nutrient availability to plants. We investigated the effect of Collembola (Protaphorura fimata Gisin) on growth and competition between of two plant species, Cirsium arvense L (creeping thistle) and Epilobium adnatum Griseb. (square-stemmed willow herb), in a laboratory experiment. Two seedlings of each plant species were planted in rhizotrons either in combination or in monoculture (intra- and interspecific competition). Interspecific competition strongly reduced total biomass of C. arvense whereas E. adnatum suffered most from intraspecific competition. Collembola neither affected the competitive relationship of the two plant species nor shoot and root biomass. Although Collembola did not affect total root biomass they influenced root morphology of both plant species. Roots grew longer and thinner and had more root tips in presence of Collembola. Root elongation is generally ascribed to the exploitation of nutrient rich patches in soil. We hypothesise that changes in root morphology in presence of Collembola are due to Collembola-mediated changes in nutrient availability and distribution. CHAPTER 2 15 2.2 Introduction Plants depend on the availability of nutrients mineralised by the decomposer community in soil. Biotic interactions in the rhizosphere of plants affect plant growth and competitiveness by a variety of mechanisms. Even though direct effects, such as root feeding, are more apparent, indirect effects of decomposers are vitally important (Setälä 1995, Wurst et al. 2003). Indirect effects of decomposers include mineralisation and distribution of nutrients, changes in activity and composition of microorganisms and modification of soil structure und root environment (Scheu and Setälä 2002, Wardle 2002). Mineralisation of nutrients in soil is mainly due to bacterial and fungal activity (Beare et al. 1992). Collembola are among the most abundant microarthropods in the rhizosphere of plants (Bardgett et al. 1993). They stimulate or reduce growth and respiration of microorganisms (van der Drift and Jansen 1977, Bakonyi 1989, Kandeler et al. 1999, Cragg and Bardgett 2001) with the direction of the effects being density-dependent (Theenhaus et al. 1999, Cole et al. 2004a). By grazing on fungi and bacteria Collembola mobilise nutrients and therefore affect plant nutrition (Teuben 1991, Lussenhop 1992, Jones 1998, Filser 2002). In fact, it has been shown that Collembola alter the content of nitrogen and phosphorus in plant shoots (Bardgett and Cook 1998, Bardgett and Chan 1999, Lussenhop and BassiRad 2005). Scheu et al. (1999) documented that in presence of Collembola both plant growth and plant shoot N content is increased. In addition, root growth is also modified by the activity of Collembola (Theenhaus et al. 1999, Cole et al. 2004a). Plant roots respond to increased nutrient availability by proliferation or an increased density and elongation of root hairs (Hodge et al. 1999). The response of plant roots depends on plant species and functional group. Theenhaus et al. (1999) demonstrated that in presence of Collembola root biomass of Trifolium repens and CHAPTER 2 16 Poa annua is decreased but the shoot/root ratio of P. annua increased. By changing plant growth and plant nutrition Collembola likely affect the competitive relationship between plant species. However, the effect of Collembola and other soil arthropods on plant competition is not clear. In a laboratory experiment Collembola increased the competitive strength of T. repens against Lolium perenne (Kreuzer et al. 2004). Schädler et al. (2004) investigated the effect of herbivorous insects on secondary plant succession of an early set-aside arable field. By applying soil and foliar insecticide, above- and belowground insects were excluded from experimental plots. Applying soil insecticide strongly affected the dominance structure of the plant community. Cirsium arvense (creeping thistle) dominated the plots with soil insecticide application, whereas in foliar insecticide treatments and in control plots Epilobium adnatum (square-stemmed willow herb) prevailed. The effects caused by insecticide applications were ascribed to herbivore insects being reduced by the insecticides. However, the observed changes in plant community may also have been due to changes in the decomposer community, e.g. in Collembola, since particularly the application of the belowground insecticide strongly reduced the density of Collembola populations and altered the dominance structure of the Collembola community (Endlweber et al. 2006). In the present study we investigated if Collembola in fact affect the competitive relationship between E. adnatum and C. arvense; these plant species were taken as model organisms to investigate the effects of Collembola on plant competition. We hypothesized that Collembola alter plant growth, root morphology and plant nutrient contents resulting in changes in the competitive strength of the two plant species. CHAPTER 2 17 2.3 Materials and Methods Rhizotrons (height 20 cm, width 15 cm, thickness 1 cm) were filled with 100 g sieved (< 1 cm) and defaunated (5 days at -21°C) soil taken from the upper 20 cm of a set- aside field near Halle (Saxony-Anhalt, Germany; cf. Schädler et al. 2004). Seedlings of C. arvense and E. adnatum were grown from seeds in pots filled with defaunated soil in the greenhouse. The seedlings were transplanted into the rhizotrons four weeks after sowing according to a replacement series design: (1) 2 seedlings of E. adnatum, (2) 1 seedling of C. arvense and 1 seedling of E. adnatum and (3) 2 seedlings of C. arvense. Two days after transplantation of the plants 60 Collembola of the euedaphic species Protaphorura fimata taken from laboratory cultures were added to half of the rhizotrons (Collembola treatments). Each treatment was replicated nine times giving a total of 54 chambers. Rhizotrons were incubated in a greenhouse (16 h light, 18°C) and arranged in a complete randomised block design. The rhizotrons were watered every other day with 5 ml deionised water. Plants were harvested by cutting at soil level after 5 weeks, when plant roots reached the bottom of the chambers. Plant height was recorded, shoots were dried at 60°C for three days and the dry weight of each shoot was determined. Dried shoots were milled in a ball mill (Retsch, Haan, Germany). Total N and C content of the plant material was analysed by an elemental analyser (Carlo Erba, Milan, Italy). Roots were washed and root length, number of root tips, root diameter and root volume were analysed using WinRHIZO (Regent Instruments Inc., Sainte-Foy, Canada). While washing the roots Collembola floating on the water surface were collected and counted. Soil respiration was measured using an automated respirometer based on electrolytic O2 microcompensation (Scheu 1992). Oxygen consumption rates at 22°C were measured every 0.5 h. Microbial basal respiration was measured as mean O2 consumption during hours 10-20 after attachment of the CHAPTER 2 18 vessels to the respirometer. Microbial biomass was assessed by measuring the maximum initial respiratory response (MIRR, µg O2 g-1 h-1) to glucose addition (substrate-induced respiration; Anderson and Domsch 1978, Beck et al. 1997). Glucose (4 mg g-1 dry wt soil) was added as an aqueous solution adjusting the soil water content to 80-90% (dry wt). MIRR was at a maximum at these glucose concentrations and moisture levels as proven in preliminary experiments. The mean of the eight lowest measurements during the first 10 h after glucose addition was taken as MIRR. The experiment was set up in a two-factorial design with the factors plant competition and Collembola (with and without). The factor plant competition analysed differences between intra- and interspecific competition for each plant species (E. adnatum and C. arvense). Individual below- and aboveground plant biomass was taken as dependent factor. Means of the factors above and belowground biomass, root length, number of root tips, root diameter, root volume and C and N content were used in treatments with intraspecific competition. The effect of Collembola on microbial biomass was analysed by single factor analyses of variance (ANOVA). Differences between means were inspected using Tukey’s honestly significant difference test. Statistical analyses were performed using the ANOVA procedure in SAS 6.12 (SAS Institute, Cary, N.C.). CHAPTER 2 19 2.4 Results 2.4.1 Collembola Collembola communities developed differently in the competition treatments. In treatments with E. adnatum and treatments with both plant species, collembolan numbers did not exceed the initially added 60 individuals. Whereas in treatments with only C. arvense collembolan numbers increased to an average of 85 individuals (SD = 24.5). Statistically, however, the two treatments did not differ significantly (F2,24=2.99, P=0.0703). 2.4.2 Plant performance Generally, shoot biomass of C. arvense exceeded that of E. adnatum. Competition affected the aboveground biomass of C. arvense and E. adnatum differently. Shoot biomass of C. arvense was significantly higher when plants grew with intraspecific competition compared to treatments with interspecific competition with E. adnatum (Table 1, Fig. 1a). Overall, root biomass as well as root length and number of root tips in E. adnatum exceeded that of C. arvense. Interspecific competition significantly reduced root biomass in C. arvense (Table 1, Fig. 1b) and caused a decrease in root length and number of root tips (Table 1, Fig. 1c, d). E. adnatum responded in the opposite way compared to treatments with interspecific competition shoot biomass decreased significantly when plants grew with intraspecific competition (Table 2, Fig. 1a). Root biomass of E. adnatum increased significantly in treatments with interspecific competition (Table 2, Fig. 1b) and this was also true for root length and number of root tips (Table 2, Fig 1c, d). CHAPTER 2 20 Table 1. Two-factor ANOVA on the effects of competition and Collembola on shoot biomass, root biomass, root volume, root diameter, root length, number of root tips, root C content, root N content and root C/N content of Cirsium arvense (n= 32). Shoot biomass Root biomass Root volume Root diameter Root length DF F P F P F P F P F P Collembola 1 5.97 0.020 1.54 0.220 3.79 0.050 0 0.988 3.11 0.090 Competition 1 0.25 0.621 12.08 0.001 5.56 0.025 0.28 0.757 10.53 0.003 Comp. x Coll. 1 2.83 0.102 0.49 0.486 0.01 0.907 0.57 0.452 0.06 0.816 Number of root tips Root C content Root N content Root C/N content DF F P F P F P F P Collembola 1 9.79 0.003 0 0.990 2.09 0.160 1.60 0.210 Competition 1 9.06 0.005 0.30 0.587 1.98 0.165 0.39 0.559 Comp. x Coll. 1 2.21 0.147 0.10 0.752 0.22 0.641 0.94 0.337 Fig. 1 intra inter A B 0 0.02 0.04 0.06 0.08 interintra sh oo t b io m as s [g ] B A E. adnatum C. arvense 0 0.01 0.02 0.03 0.04 B A A B ro ot b io m as s [g ] intra interinterintra E. adnatum C. arvense(a) (b) A B 0 100 200 300 400 500 600 700 B A intra interinterintra E. adnatum C. arvense(d) 0 100 200 300 400 R oo tl en gt h [c m ] intra interinterintra E. adnatum C. arvense B B A A (c) m ea n nu m be ro f r oo tt ip s pe r p la nt Fig. 1 intra inter A B 0 0.02 0.04 0.06 0.08 interintra sh oo t b io m as s [g ] B A E. adnatum C. arvense 0 0.01 0.02 0.03 0.04 B A A B ro ot b io m as s [g ] intra interinterintra E. adnatum C. arvense(a) (b) A B 0 100 200 300 400 500 600 700 B A intra interinterintra E. adnatum C. arvense(d) 0 100 200 300 400 R oo tl en gt h [c m ] intra interinterintra E. adnatum C. arvense B B A A (c) m ea n nu m be ro f r oo tt ip s pe r p la nt intra inter A B 0 0.02 0.04 0.06 0.08 interintra sh oo t b io m as s [g ] B A E. adnatum C. arvense 0 0.01 0.02 0.03 0.04 B A A B ro ot b io m as s [g ] intra interinterintra E. adnatum C. arvense(a) (b) A B 0 100 200 300 400 500 600 700 B A intra interinterintra E. adnatum C. arvense(d) 0 100 200 300 400 R oo tl en gt h [c m ] intra interinterintra E. adnatum C. arvense B B A A (c) m ea n nu m be ro f r oo tt ip s pe r p la nt Figure 1. Effect of inter- and intraspecific competition on shoot biomass (a), root biomass (b), root length (c) and number of root tips (d) in C. arvense and E. adnatum. Error bars are one standard error from the mean. Bars with the same letter are not significantly different (Tukey’s honestly significant difference, P<0.05) CHAPTER 2 21 Collembola generally did not affect shoot and root biomass of C. arvense (Table 1) and E. adnatum (Table 2). However, they strongly affected the structure of the root system of both plant species. Overall, Collembola reduced the diameter and volume of roots in E. adnatum (Table 2, Fig. 2a, b). In contrast, root length was significantly increased (Table 2, Fig. 3). They also increased the number of root tips (Table 2, Fig. 4). In contrast to E. adnatum, Collembola neither affected root length nor root volume of C. arvense. However, similar to E. adnatum presence of Collembola reduced the diameter of roots in C. arvense (Table 1, Fig. 2a) and increased the number of root tips (Table 1, Fig. 4). There was generally no significant interaction between Collembola and plant competition on any of the plant response variables studied (Table 1, 2). Table 2. Two-factor ANOVA on the effects of competition and Collembola on shoot biomass, root biomass, root volume, root diameter, root length, number of root tips, root C content, root N content and root C/N content of Epilobium adnatum (n= 32). Shoot biomass Root biomass Root volume Root diameter Root length DF F P F P F P F P F P Collembola 1 7.75 0.009 1.30 0.260 21.30 <0.0001 28.41 <0.0001 5.42 0.026 Competition 1 0.44 0.511 4.70 0.035 3.30 0.0790 0.03 0.8570 8.47 0.007 Comp. x Coll. 1 0.74 0.395 1.67 0.208 1.24 0.2740 2.30 0.1360 3.24 0.081 Number of root tips Root C content Root N content Root C/N content DF F P F P F P F P Collembola 1 9.36 0.005 0.02 0.878 0.65 0.410 0.95 0.335 Competition 1 4.23 0.048 7.15 0.010 0.13 0.722 4.52 0.039 Comp. x Coll. 1 1.08 0.308 1.42 0.239 0.10 0.658 3.90 0.060 Presence of Collembola did not cause any changes in microbial basal respiration (overall mean 2.36 µg O2 h-1 g-1 dry weight) nor in microbial biomass (overall mean 249.3 µg Cmic g-1 dry weight). Although Collembola and plant competition affected plant biomass and root performance plant tissue nitrogen and carbon concentration were not affected except for root C content of E. adnatum which was significantly CHAPTER 2 22 increased when grown together with C. arvense (Table 2). The C content of E. adnatum was increased from 34.68% when grown as single species to 36.74% in interspecific competition treatments; leading to an increased C/N ratio in E. adnatum roots when grown in competition with C. arvense (from 27.59% to 29.59%; Table 2). ro ot vo lu m e [c m ²] 0 0.2 0.4 0.6 0.8 - Coll + Coll E. adnatum C. arvense - Coll + Coll (b) A A A B Fig. 2 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 ro ot di am et er [m m ] - Coll + Coll E. adnatum C. arvense - Coll + Coll (a) B A B A Fig. 3 0 100 200 300 400 to ta l r oo tl en gt h [c m ] - Coll + Coll E. adnatum C. arvense A B + Coll A A - Coll Fig. 4 0 200 400 600 800 m ea n nu m be ro f r oo tt ip s pe r p la nt - Coll + Coll E. adnatum C. arvense - Coll + Colll B A B A Figure 3. Effect of Collembola on total root length in C. arvense and E. adnatum. Error bars are one standard error from the mean. Bars with the same letter are not significantly different (Tukey’s honestly significant difference, P<0.05) ro ot vo lu m e [c m ²] 0 0.2 0.4 0.6 0.8 - Coll + Coll E. adnatum C. arvense - Coll + Coll (b) A A A B ro ot vo lu m e [c m ²] 0 0.2 0.4 0.6 0.8 - Coll + Coll E. adnatum C. arvense - Coll + Coll (b) AA AA AA BB Fig. 2 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 ro ot di am et er [m m ] - Coll + Coll E. adnatum C. arvense - Coll + Coll (a) BB AA BB AA Fig. 3 0 100 200 300 400 to ta l r oo tl en gt h [c m ] - Coll + Coll E. adnatum C. arvense AA BB + Coll AA A - Coll A - Coll Fig. 4 0 200 400 600 800 m ea n nu m be ro f r oo tt ip s pe r p la nt - Coll + Coll E. adnatum C. arvense - Coll + Colll B A B A Fig. 4 0 200 400 600 800 m ea n nu m be ro f r oo tt ip s pe r p la nt - Coll + Coll E. adnatum C. arvense - Coll + Colll BB AA BB AA Figure 3. Effect of Collembola on total root length in C. arvense and E. adnatum. Error bars are one standard error from the mean. Bars with the same letter are not significantly different (Tukey’s honestly significant difference, P<0.05) Figure 2. Effect of Collembola on root diameter (a) and root volume (b) in C. arvense and E. adnatum. Error bars are one standard error from the mean. Bars with the same letter are not significantly different (Tukey’s honestly significant difference, P<0.05) Figure 4. Effect of Collembola on number of root tips in C. arvense and E. adnatum. Error bars are one standard error from the mean. Bars with the same letter are not significantly different (Tukey’s honestly significant difference, P<0.05) CHAPTER 2 23 2.5 Discussion Competition between plants for nutrients in soil is an important factor forming plant communities (Aerts 1999). Intra- and interspecific competition of plant roots influences root proliferation and structure of root systems (Gersani et al. 1998). Gersani et al. (2001) and Maina et al. (2002) documented that plants react to inter- and intraspecific competition by the production of more roots and less yield. However, the response of plants to inter- and intraspecific competition may vary with plant species. Schädler et al. (2004) suggested that C. arvense and E. adnatum, two common plant species on arable land and fallow fields, compete for resources in early successional stages after cessation of cultivation. In the present study the effect of inter- and intraspecific competition on plant growth differed between the two plant species. Intraspecific competition decreased root and shoot biomass in E. adnatum whereas C. arvense suffered most from interspecific competition suggesting that E. adnatum is the stronger interspecific competitor (Ang et al. 1995). Besides competition for space and light plants compete mainly for nutrients in soil. Belowground competition influences the root structure of the plants (Volis and Shani 2000) but the extent depends on root architecture of the competing plant species (Rubio et al. 2001). In contrast to our hypothesis that Collembola affect the competitive relationship between E. adnatum and C. arvense, results of the present experiment suggest that the competitive relationship between the two plant species is independent of the presence of Collembola. Collembola neither affected shoot nor root biomass in the two plant species which is in contrast to previous studies reporting an increase in plant biomass in presence of Collembola (Scheu et al. 1999, Kreuzer et al. 2004, Lussenhop and BassiriRad 2005). These contrasting results might be due to variable responses of plant species and plant developmental stages to the presence of CHAPTER 2 24 Collembola. The effect of Collembola on plant growth varies with plant functional group (Partsch et al. 2006). Presence of different Collembola species with different functional characteristics might be more important than Collembola density (Cragg and Bardgett 2001, Cole et al. 2004a). Previous experiments documenting changes in root and shoot biomass in presence of Collembola lasted for 8-12 weeks (Scheu et al. 1999, Kreuzer et al. 2004, Gormsen et al. 2004, Lussenhop and BassiriRad 2005, Partsch et al. 2006), whereas experiments which did not find any changes in plant growth due to the presence of Collembola were running for about 3-5 weeks (Larsen and Jakobsen 1996). In the present study though Collembola did not affect total root biomass they affected root morphology of both plant species. Roots generally were longer and thinner and developed more root tips in presence of Collembola. This was more pronounced in E. adnatum than in C. arvense. A major determinant of the structure of the root system of plants is the availability and distribution of nutrients in soil. Roots respond to nutrient rich patches by proliferation and growth towards these patches. Proliferation is primarily triggered by the availability of nitrogen and includes a variety of responses comprising increased production of root tissue and production of longer roots (van Vuuren et al. 1996, Hodge et al. 1999, Hodge et al. 2000). The ability of roots to grow to nutrient rich patches differs between species and is influenced by competition between plants for resources in soil (Hutchings et al. 2003). Collembola may affect root structure by influencing the nutrient availability and spatial heterogeneity of nutrients in soil (McGonigle 1995). Collembola form discrete nutrient rich patches through excretion and faecal pellet deposition (Petersen 2000, Sjursen and Holmstrup 2004). As indicated by increased root elongation and number of root tips in E. adnatum and C. arvense plant roots exploited these nutrient rich patches by growing longer. Root elongation and root branching are typical reactions to patchily CHAPTER 2 25 distributed nutrient rich sources (Linkohr et al. 2002, Mantelin and Touraine 2004). Furthermore, Collembola affect the availability of nutrients by modification of microbial biomass and activity. It has been demonstrated that mineralisation of nitrogen in presence of Collembola and other soil microarthropods is increased (Bardgett and Chan 1999). Plants benefit from the increased availability of nitrogen resulting in increased plant growth and tissue nitrogen concentration (Kreuzer et al. 2004, Lussenhop and BassiRad 2005). In the present study no effects of Collembola on microbial biomass and activity could be detected which is in contrast to previous experiments (Hanlon and Anderson 1979, Bakonyi 1989, Teuben 1991, Kandeler et al. 1999). It is known that the effect of Collembola on microorganisms depends on microarthropod density and on species composition (Cragg and Bardgett 2001, Cole et al. 2004a, Cole et al. 2004b). Microbial biomass may have remained unaffected since Collembola density was low compared to other studies (Scheu et al. 1999, Kreuzer et al. 2004, Partsch et al. 2006). Root elongation and an increase in the number of root tips suggest increased availability of nitrate (Zhang and Forde 2000, Mantelin and Touraine 2004). In fact, Cragg and Bardgett (2001) found enhanced leaching of nitrate in presence of Collembola and attributed this to an increased activity of nitrifying bacteria. Further studies are necessary combining the analysis of the structure of the plant root system and the composition of microbial communities in the rhizosphere as affected by the presence of Collembola. CHAPTER 2 26 2.6 Conclusions Overall, results of the present study documented that despite plant biomass production and plant competition remain unaffected, Collembola altered the structure of the root system of plants and root resource exploitation. Considering that the effect of Collembola varies with plant species and soil conditions these are likely to alter plant growth and plant competition in the field. Therefore, experiments using insecticides to exclude herbivore insects for investigating their effect on plant communities have to consider that the observed effects in fact at least in part might be due to changes in the decomposer community. In the insecticide treatments of the studied oldfield community decomposers, such as collembolans, might have contributed to the observed changes in dominance between C. arvense and E. adnatum. CHAPTER 3 27 CHAPTER 3 INTERACTIONS BETWEEN MYCORRHIZAL FUNGI AND COLLEMBOLA: EFFECTS ON ROOT STRUCTURE OF COMPETING PLANT SPECIES 3.1 Abstract Mycorrhizal fungi influence plant nutrition and therefore likely modify competition between plants. By affecting mycorrhiza formation and nutrient availability of plants, Collembola may influence competitive interactions of plant roots. We investigated the effect of Collembola (Protaphorura fimata Gisin), a mycorrhizal fungus (Glomus intraradices Schenck and Smith), and their interaction on plant growth and root structure of two plant species, Lolium perenne L (perennial ryegrass) and Trifolium repens L (white clover). In a laboratory experiment two individuals of each plant species were grown either in monoculture or in competition to the respective other plant species. Overall, L. perenne built up more biomass than T. repens. The clover competed poorly with grass, whereas the L. perenne grew less in presence of conspecifics. In particular, presence of conspecifics in the grass and presence of grass in clover reduced shoot and root biomass, root length, number of root tips and root volume. Collembola reduced shoot biomass in L. perenne, enhanced root length and number of root tips, but reduced root diameter and volume. Effects of Collembola on T. repens were less pronounced but Collembola enhanced root length and number of root tips. In contrast to our hypothesis, changes in plant biomass and root structure in the presence of Collembola were not associated with a reduction in mycorrhizal formation. Presumably, Collembola affected root structure via changes in the amount of nutrients available and their spatial distribution. CHAPTER 3 28 3.2 Introduction One of the most important systems affecting plant nutrition is the symbiosis of plant roots with mycorrhizal fungi (Smith and Read 1997). Mycorrhizal fungi facilitate plant nutrient uptake, in particular that of phosphate, but also that of other nutrients, such as zinc and copper. Additionally, the symbiosis can provide the plant with inorganic nitrogen (Javelle et al. 1999, Hawkins et al. 2000, Hawkins and George 2001). The functioning of mycorrhizas, however, is affected by other biota. Mycorrhizal fungi are imbedded in a complex food web of decomposer invertebrates including soil arthropods, such as Collembola. Collembola graze on hyphae and spores of arbuscular mycorrhizal fungi (Moore et al. 1987, Bakonyi et al. 2002) and this may significantly affect plant growth (Kaiser and Lussenhop 1991, Gange 2000, Kreuzer et al. 2004). The effect of Collembola on mycorrhizal functioning has been shown to be density dependent, with high Collembola densities hampering but low densities increasing mycorrhizal nutrient transfer to plants (Ek et al. 1994). Reduced mycorrhizal functioning might be due to lower infection of roots with mycorrhizal fungi (Lussenhop 1996) caused by feeding on spores and hyphae (Klironomos and Ursic 1998, Bakonyi et al. 2002). The increase in mycorrhizal functioning at low densities of Collembola is likely due to a stimulation of hyphal growth and functioning (Kandeler et al. 1999, Gange 2000, Cragg and Bardgett 2001). In addition, Collembola might also beneficially affect ectomycorrhizal fungi and increase root infection by transporting spores. It has been shown that spores of more than 100 fungal species adhere to the body surface of Onychiurus subtenuis (Visser et al. 1987). Most Collembola species preferentially feed on saprotrophic rather than mycorrhizal fungi (Klironomos and Ursic 1998, Schreiner and Bethlenfalvay 2003). Selective grazing of Collembola on fungi may result in changes in the structure of fungal CHAPTER 3 29 communities, e.g. by decreasing the competitive strength of saprotrophic fungi and increasing that of mycorrhizal fungi (Sabatini and Innocenti 2000, Tiunov and Scheu 2005). Changes in the structure of the soil fungal community likely result in changes in plant nutrition and therefore affect plant growth. Furthermore, Collembola affect plant growth by increasing nutrient availability by feeding on bacteria and fungi, and mobilising microbial nutrient pools (Lussenhop 1992, Theenhaus et al. 1999, Filser 2002, Cole et al. 2004a). Plant roots respond to modified nutrient availability by proliferation and elongation even though shoot and root biomass may remain unaffected (Hodge et al. 1999, Endlweber and Scheu 2006). By changing plant nutrition, root growth and root structure, Collembola not only affect plant growth but likely also plant competition. In the laboratory Collembola indeed increased the competitive strength of Trifolium repens L against Lolium perenne L (Kreuzer et al. 2004). The present experiment builds on these results by investigating if these changes are mediated by mycorrhiza. We hypothesise that changes in plant biomass and root structure are due to a reduction in mycorrhizal formation by Collembola. Therefore, we investigated effects of Collembola on arbuscular mycorrhizal fungus infection, plant nutrient uptake and root morphology of L. perenne and T. repens growing in monoculture and in combination of both plant species. 3.3 Materials and Methods The experiment was conducted in a temperature controlled greenhouse (16 h light, 18°C). Rhizotrons (height 35 cm, width 15 cm, thickness 1 cm) were filled with 280 g soil taken from the upper 20 cm of a set-aside field (early successional stage) in Bad Lauchstädt near Halle (Saxony-Anhalt, Germany). The soil is a Chernosem with an average pH of 7.14 and an average content of 10.19 μg PO- 4-P/g soil. Carbonate- CHAPTER 3 30 extractable phosphate was extracted as reported by Olsen and Sommers (1982). Average ammonium and nitrate contents were 0.43 μg NH4 +/ g soil and 0.96 μg NO3 - /g soil respectively. Mineral N was extracted from sub-samples and was determined as reported by Keeney and Nelson (1982). The soil was sieved (1 cm mesh) and autoclaved at 120°C for 2 h for defaunation and elimination of mycorrhizal fungi. It was stored for 3 days at 15°C after autoclaving and then filled into the experimental containers. Fresh soil (172g dry weight) was suspended in 200 ml distilled water to re-inoculate the soil with microorganisms. The suspension (150 ml) was filtered through 25 µm gauze to exclude mycorrhizal fungus spores and made up to 750 ml with distilled water. Soil suspension (12 ml) was evenly distributed over the soil of each rhizotron. Seven-day-old seedlings of L. perenne and T. repens were transplanted into the rhizotrons to establish the following treatments in a replacement series design: (1) 2 seedlings of L. perenne, (2) 1 seedling of L. perenne and 1 seedling of T. repens, (3) 2 seedlings of T. repens. Each treatment was inoculated with or without mycorrhizal fungi, and with and without Collembola. Each treatment was replicated five times giving a total of 60 rhizotrons. Mycorrhizal fungus (14 g) as spores and hyphae of Glomus intraradices Schenck and Smith (Dr. C. Grotkass, Institut für Pflanzenkultur, Schnega, Germany), was evenly spread over the soil in each rhizotron. Non- mycorrhizal treatments received the same amount of inoculum which had been autoclaved (120°C, 2 h). In order to control for potential effects caused by microorganisms other than mycorrhizal fungi in the inoculum 12 ml of a filtrate of the inoculum were added to each experimental container. The filtrate was prepared by suspending 100 g of the inoculum in 800 ml distilled water and filtered through 25 µm gauze. Half of the rhizotrons received 100 collembolans of the euedaphic species CHAPTER 3 31 Protaphorura fimata Gisin. The rhizotrons were watered with 15 ml distilled water every other day through the experiment. Plants were harvested after eight weeks. Shoots were dried at 60°C for three days and weighed. Dried shoots were milled in a ball mill (Retsch, Haan, Germany) and the shoot C and N contents were analysed by an elemental analyser (Carlo Erba, Milan, Italy). Plant roots were washed and scanned. Images were analysed in terms of root length, number of root tips, root diameter and root volume using WinRHIZO (Regent Instruments Inc., Sainte-Foy, Canada). Collembola floating on the water surface during root washing were collected and counted. The roots were weighed and a subsample was bleached by boiling in 1 N KOH. Then, the roots were dyed in 10 ml 1 N HCl mixed with two drops ink (Quink, Parker Permanent Blue, Germany) and bleached in a mixture of 10 ml lactic acid and 10 ml distilled water. Colonisation of roots by mycorrhizal fungi was analysed using the gridline intersection method (Giovannetti and Mosse 1980). To determine root biomass, roots were dried at 60°C for three days and weighed. The experiment was set up in a complete factorial design with three factors: plant combination (con- and heterospecifics), Collembola (with and without) and mycorrhiza (with and without). Effects of these factors on above and belowground biomass, root length, number of root tips, root diameter, root volume and C and N content were analysed by three factorial ANOVA. In treatments with conspecific competitors (monocultures) means of the dependent variables of the two plant individuals per rhizotron were used for the analyses. Collembola density and mycorrhizal fungus inoculation were analysed by two factor ANOVA with mycorrhiza and plant competition as independent variables. Differences between means were inspected using Tukey’s honestly significant difference test. Statistical analyses were performed using the ANOVA procedure in SAS 6.12 (SAS Institute, Cary, N.C.). CHAPTER 3 32 3.4 Results 3.4.1 Plant combination The shoot yield of L. perenne was significantly higher when grown in combination with T. repens compared to when grown in monoculture (Table 1, Fig. 1a). Increased shoot biomass was not associated with changes in root biomass. However, roots of L. perenne were significantly longer (Table 1, Fig. 2a) and the number of root tips was enhanced (Table 1, Fig. 3a) when grown with T. repens compared to monoculture. Although root biomass and the diameter of roots (Table1, Fig. 4) did not differ between the treatments, root volume of L. perenne was significantly increased Figure 1. Effect of presence of con- and hete when grown with T. repens (Table 1). rospecific competitors, Collembola and mycorrhization on shoot biomass of Lolium perenne and Trifolium repens respectively. Bars sharing the same letter are not significantly different (Tukey’s honestly significant difference, P<0.05) Fig. 1 0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 A B A A - Myc + Myc- Coll + Coll A het B con sh oo tb io m as s [g ] 0 0.01 0.02 0.03 0.04 0.05 0.06 A A sh oo tb io m as s [g ] - Myc + Myc- Coll + Coll B het A con A A (a) (b) Lolium perenne Trifolium repens Fig. 1 0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 A B A A - Myc + Myc- Coll + Coll A het B con sh oo tb io m as s [g ] 0 0.01 0.02 0.03 0.04 0.05 0.06 A A sh oo tb io m as s [g ] - Myc + Myc- Coll + Coll B het A con A A (a) (b) Lolium perenne Trifolium repens CHAPTER 3 33 The shoot biomass of T. repens grown with conspecifics was significantly higher than when grown with L. perenne (Table 2, Fig. 1b). When grown with L. perenne root : 9.831). Figure 2. Effect of presence of con- and heterospeci gth of Lolium perenne and Trifolium repens respectively. Bars sharing the same letter are cantly different (Tukey’s honestly significant difference, P<0.05) on root len not signifi biomass (Table 2) and the length of roots (Table 2, Fig. 2b) of T. repens were significantly reduced. However, root diameter (Table 2, Fig. 4), root volume and number of root tips remained unaffected (Table 2). Shoot C/N ratio in T. repens and L. perenne were not significantly affected by plant combination (average: T. repens: 15.389, L. perenne Fig. 2 Lolium perenne fic competitors, Collembola and mycorrhization 0 200 400 600 800 A B A A - Myc + Myc- Coll + Coll B het A con to ta l ro ot le ng th [c m ] 0 50 100 150 200 250 300 A B A B - Myc + Myc- Coll + Coll B het A con (a) (b) to ta l ro ot le ng th [c m ] Fig. 2 Lolium perenne Trifolium repens 0 200 400 600 800 A B A A - Myc + Myc- Coll + Coll B het A con to ta l ro ot le ng th [c m ] 0 50 100 150 200 250 300 (a) (b) to ta l ro ot le ng th [c m ] Trifolium repens A con B het B A A B - Myc + Myc- Coll + Coll HAPTER 3 34 Table 1. F-values of a three-factor ANOVA on the effects of Collembola, presence of con-/heterospecific competitors and mycorrhiza on shoot biomass, root biomass, root volume, root diameter, root length and number of root tips of Lolium perenne (n= 5). Shoot biomass Root biomass Root volume Root diameter Root length Root tips df F P F P F P F P F P F P Collembola (Coll.) 1 5.72 0.0228 3.22 0.0823 131.86 <0.0001 42.18 <0.0001 104.60 <0.0001 5.72 0.0228 Plant combination (Com.) 1 22.59 <0.0001 0.05 0.8201 23.70 <0.0001 0.14 0.7117 23.93 <0.0001 22.59 <0.0001 Mycorrhiza (Myc.) 1 0.19 0.6629 0.02 0.8804 0.98 0.3298 0.12 0.7317 2.77 0.1061 0.19 0.6629 Coll. x Com. 1 2.80 0.1043 1.94 0.1728 32.36 <0.0001 9.69 0.0039 20.49 <0.0001 2.80 0.1043 Coll. x Myc. 1 0.85 0.3635 0.05 0.8201 0.44 0.5114 0.14 0.7090 0.04 0.8417 0.85 0.3635 Com. x Myc. 1 0.29 0.5945 0.21 0.6522 0.24 0.6266 0.35 0.5597 1.45 0.2379 0.29 0.5945 Coll. x Com. x Myc. 1 0.05 0.8164 0.18 0.6708 2.63 0.1146 1.95 0.1724 4.17 0.0494 0.05 0.8164 df = degrees of freedom Table 2. F-values of a three-factor ANOVA on the effects of Collembola, presence of con-/heterospecific competitors and mycorrhiza on shoot biomass, root biomass, root volume, root diameter, root length and number of root tips of Trifolium repens (n= 5). Shoot biomass Root biomass Root volume Root diameter Root length Root tips df F P F P F P F P F P F P Collembola (Coll.) 1 0.55 0.4625 0.31 0.5795 2.31 0.1396 0.79 0.3801 6.98 0.0131 4.39 0.0450 Plant combination (Com.) 1 24.93 <0.0001 4.85 0.0347 0.47 0.4966 0.08 0.7755 4.96 0.0338 2.01 0.1672 Mycorrhiza (Myc.) 1 0.51 0.4784 1.31 0.2601 3.45 0.0733 5.30 0.0287 5.45 0.0267 7.13 0.0123 Coll. x Com. 1 1.45 0.2381 0.06 0.8051 1.88 0.1809 0.73 0.3990 3.28 0.0806 0.41 0.5249 Coll. x Myc. 1 1.05 0.3131 0.00 1.0000 0.70 0.4099 1.30 0.2644 0.30 0.5905 0.01 0.9124 Com. x Myc. 1 2.07 0.1599 1.15 0.2919 0.40 0.5306 0.06 0.8112 0.15 0.6975 0.73 0.3985 Coll. x Com. x Myc. 1 0.10 0.7527 1.14 0.2925 4.45 0.0436 2.29 0.1412 8.78 0.0060 6.61 0.0155 df = degrees of freedom C CHAPTER 3 35 3.4.2 Effects of mycorrhiza Inoculation with G. intraradices mycorrhiza affected neither below- nor aboveground biomass of L. perenne (Table 1, Fig. 1a). Furthermore, inoculation of plant roots with mycorrhizal fungi did not affect root length, root volume and root diameter or the number of root tips (Fig. 2a, 3a, 4a). Fig. 3 0 100 200 300 400 A B A B nu m be ro f r oo tt ip s - Myc + Myc- Coll + Coll A het A con A B 0 400 800 1200 1600 2000 - Myc + Myc- Coll + Coll A het B con AA nu m be ro f r oo tt ip s (a) (b) Lolium perenne Trifolium repens Fig. 3 0 100 200 300 400 A B A B nu m be ro f r oo tt ip s - Myc + Myc- Coll + Coll A het A con A B 0 400 800 1200 1600 2000 - Myc + Myc- Coll + Coll A het B con AA nu m be ro f r oo tt ip s (a) (b) Lolium perenne Trifolium repens Figure 3. Effect of presence of con- and heterospecific competitors, Collembola and mycorrhization on root diameter of Lolium perenne and Trifolium repens respectively. Bars sharing the same letter are not significantly different (Tukey’s honestly significant difference, P<0.05) CHAPTER 3 36 As in L. perenne, inoculation with the mycorrhizal fungus did not affect shoot or root biomass of T. repens, but significantly reduced root length (Table 1, 2, Fig. 2b), root diameter and the number of root tips (Table 2, Fig. 3b, 4b). Furthermore, the extent of mycorrhizal inoculation of roots of T. repens grown with L. perenne significantly exceeded that when grown with conspecifics (F5,24=4.74, P=0.0410). The average colonisation of roots of L. perenne by G. intraradices (86.66% of root length) generally exceeded that of T. repens (67.97%). Inoculation with mycorrhizal fungi affected neither C/N ratio of T. repens nor that of L. perenne (average: T. Figure 4. Effect of presence of con- and he repens: 15.336, L. perenne: 9.809). terospecific competitors, Collembola and mycorrhization on root number of root tips Lolium perenne and Trifolium repens respectively. Bars sharing the same A B 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 - Myc + Myc- Coll + Collinter AA intra A A ro ot di am et er [m m ] A B 0 0.2 0.4 0.6 0.8 1.0 - Myc + Myc- Coll + Collinter A intra A A A ro ot di am et er [m m ] Lolium perenne Trifolium repens A B 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 - Myc + Myc- Coll + Collinterinter AA intra A intra A A ro ot di am et er [m m ] A B 0 0.2 0.4 0.6 0.8 1.0 - Myc + Myc- Coll + Collinter A intra A A A ro ot di am et er [m m ] Lolium perenne Trifolium repens letter are not significantly different (Tukey’s honestly significant difference, P<0.05) CHAPTER 3 37 3.4.3 Effects of Collembola hoot but not root biomass of L. perenne (Table 1, Fig. 1a). However, Collembola significantly increased root length (Table 1, Fig. 2a) and the number of root tips (Table 1, Fig. 3a), but decreased root volume and root diameter (Table 1, Fig. 4a). Collembola did not affect above- and belowground biomass, root volume and root diameter of T. repens. However, they did enhance the length of roots of T. repens, but reduced the number of root tips (Table 2, Fig. 1a, 2a, 3a, and 4a). In contrast, Collembola significantly reduced the colonisation of roots of T. repens by mycorrhizal fungi by about 20% (F =5.23 P=0.0327), but did not affect mycorrhizal infection of roots of L. perenne. Collembola generally did not affect the C/N ratio of the two plant species (average: T. repens: 15.338, L. perenne: 9.809). Inoculation of plant roots with G. intraradices generally did not affect Collembola density, but the number of Collembola significantly differed between the competition treatments (F =13.79, P=0.0006), with an average of 162 individuals per rhizotron in T. repens monocultures and an approximately three times higher density in L. perenne monocultures and in combinations of T. repens and L. perenne (average of 403 and 435 individuals per rhizotron, respectively). Collembola significantly reduced s 3,26 5,24 CHAPTER 3 38 3.5 Discussion ination mportant factors structuring plant communities. Plant responses may differ between intra- and interspecific competition depending on plant species and plant functional group (Gersani et al. 2001, Maina et al. 2002). Plant competitiveness varies with plant root structure and root foraging strategy (Lodge 2000, Rajaniemi and Reynolds 2004). Hence, plant competitiveness likely increases with the growth rate of roots and the extension of the root system (Aerts 1999). In the present experiment L. perenne grew faster and built up significantly more biomass than T. repens. The presence of L. perenne was associated with a reduced biomass of T. repens compared to monoculture treatments. This is consistent with findings of other studies demonstrating a competitive superiority of ryegrass over clover (Munoz and Weaver 1999, Lucero et al. 1999). Strong competitiveness of L. perenne likely is due to the ramified root system which is characteristic for grasses and allows effective uptake of nutrients (Stone et al. 1998). Furthermore, in soils with high N availability the competitive advantage of nitrogen-fixing legumes is abrogated. Autoclaving soil, as done in the present experiment to eliminate mycorrhiza, mobilises nutrients, especially N, and may have contributed to the low competitiveness of T. repens in our experiment. This is supported by the low number of nodules observed for T. repens. The different responses of the two plant species to monoculture and the combined treatment are probably due to the different growth rates of both plant species. The grass had a higher growth rate than the clover. Therefore, competition between conspecifics of L. perenne was higher in the limited space of the rhizotrons compared to monocultures of T. repens. Furthermore the grass outcompeted clover in the combined treatments. 3.5.1 Plant comb Competition is one of the most i CHAPTER 3 39 In addition to shoot biomass, presence of con- and heterospecific competitors also affected root structure; similar results have been documented (Gersani 1998). Intra- 3.5.2 Mycorrhiza The response of plants to inoculation with AM fungi species varies with the s (Joner and Leyval 2001, Rogers et al. 2001, Klironomos 2003). In ot system (Smith and Read 1997, Harrison 1997, van der Heijden 2004). Colonisation of roots by mycorrhizal fungi and interspecific competition often results in an increase in root biomass (Maina et al. 2002, O’Brien et al. 2005). Generally, competition between plant roots is more intense among plants with similar root morphology (Rubio et al. 2001). In the present experiment, L. perenne produced longer roots, more root tips and an enhanced root volume when competing with T. repens as compared to when grown with conspecifics. In contrast, T. repens produced longer roots when grown in monoculture as compared to when grown in competition with L. perenne. Root proliferation is strongly affected by the availability of nutrients and allows plants to exploit resources to the disadvantage of the competitor (Hodge et al. 1999, Gersani et al. 2001). There is evidence that plant species are able to differentiate conspecific and heterospecific roots and adjust the response of the root system accordingly (Huber-Sannwald et al. 1996). mycorrhizal fungu the present experiment root colonisation with mycorrhizal fungi in L. perenne exceeded that in T. repens. Nevertheless, plant growth and root morphology in L. perenne were little affected by mycorrhiza whereas in T. repens mycorrhiza reduced root length, number of root tips and root diameter. Mycorrhizal fungi allow plants to reduce investment into roots since mycorrhizal hyphae compensate for reduced extension of the ro CHAPTER 3 40 therefore likely affects competition of plant species (van der Heijden et al. 2003, Smith et al. 1999). Indeed, in previous experiments mycorrhizal fungi increased the competitive strength of clover against ryegrass (Hamel et al. 1992, Joner and Leyval 2001). This might have been due to higher colonisation of roots by mycorrhizal fungi of the legume compared to the grass. However, in the present experiment mycorrhizal fungi did not increase the competitiveness of clover. This might have been due to the higher colonisation by mycorrhizal fungi of L. perenne roots compared to roots of T. repens. Colonisation by mycorrhizal fungi is probably correlated with the plant nutrient status. Blanke et al. (2005) demonstrated a negative correlation between root colonisation of Artemisia vulgaris by AMF and tissue N concentration. Therefore, differences in colonisation by mycorrhizal fungi are possibly due to different nutrient contents. 3.5.3 Collembola Collembola may stimulate or reduce plant nutrition and growth (Harris and Boerner 1990, Bardgett and Chan 1999, Scheu et al. 1999, Lussenhop and BassiriRad 2005). bola may significantly affect root growth without affecting shoot In addition, Collem growth (Scheu et al. 1999, Endlweber and Scheu 2006). In the present experiment, Collembola increased root length and number of root tips in both plant species but reduced shoot biomass, root volume and root diameter in L. perenne. The reduction in shoot biomass of L. perenne was more pronounced when grown with T. repens suggesting that Collembola reduced the competitive superiority of L. perenne over T. repens. These findings and previous experiments (Kreuzer et al. 2004, Partsch et al. 2006) suggest that Collembola generally increase the competitive strength of legumes against grasses. The increase in the competitive strength of legumes may CHAPTER 3 41 be caused by increased nodule occupancy in legumes in the presence of Collembola (Lussenhop 1993). Increased root elongation and number of root tips likely reflect an increase in the availability of nitrate (Zhang and Forde 2000, Mantelin and Touraine 2004). Collembola may increase N availability in the rhizosphere via enhancing microbial N hytic fungi (cf. Salamon et al. 2004, Sung et al. 2006). mineralisation and by forming nutrient rich patches through excretion (Bardgett and Chan 1999, Petersen 2000, Sjursen and Holmstrup 2004). Presumably, in the present experiment mineral N made available by Collembola at microsites in soil, such as droppings of excreta, was nitrified quickly and this stimulated root elongation and branching. However, the potentially increased availability of N by Collembola was not reflected by shoot N concentration. In fact, the reduction of shoot biomass by Collembola contradicts the assumption that Collembola enhanced nutrient availability to plants. This reduction might have been due to a decline in nutrients provided by mycorrhizas. Collembola have been shown to alter mycorrhization of plant roots with the effect being density dependent (Ek et al. 1994, Lussenhop 1996, Bakonyi et al. 2002). In the present experiment Collembola reduced the colonisation of roots by mycorrhiza of T. repens which likely was caused by grazing on mycorrhizal hyphae. Surprisingly, however, Collembola did not significantly affect root colonisation by mycorrhiza in L. perenne although mycorrhizal colonisation of roots in L. perenne exceeded that in T. repens. Collembola density was significantly higher in treatments with ryegrass suggesting that they benefited from high root biomass and associated high root exudates and increased biomass of saprop Milcu et al. (2006) found Collembola density to be reduced in the presence of legumes, whereas it was increased in the presence of grasses. CHAPTER 3 42 3.6 Conclusions Presence of Collembola alters root structure with longer and thinner roots and erefore likely affects plant resource exploitation. Although plant C and N content a declined shoot biomass. Overall, results of the present experiment and previous studies suggest that the effect of Collembola depends on plant species with grasses being more vulnerable than legumes. Therefore, Collembola likely reduce the competitive superiority of grasses over legumes. The Collembola-mediated reduction in shoot biomass and changes in root structure were not related to changes in mycorrhizal fungus colonisation of roots. Hence, the effect of Collembola on root morphology and shoot biomass presumably is not caused by affecting plant-mycorrhiza interrelationships, but possibly by direct grazing on roots and on saprotrophic fungi. th remained unaffected presence of Collembol CHAPTER 4 43 CHAPTER 4 DIETARY ROUTING IN COLLEMBOLA: DETERMINING COLLEMBOLA FEEDING PREFERENCES BY STABLE ISOTOPE AND COMPOUND SPECIFIC FATTY ACID ANALYSIS 4.1 Abstract Collembola are abundant and ubiquitous soil decomposers, being particularly active in the rhizosphere of plants where they are assumed to be attracted by high microbial activity and biomass. While feeding on root associated microorganisms or organic matter they may damage and ingest plant roots e.g. particularly root hairs and fine roots. Employing stable isotope analysis and compound specific 13C analysis of fatty acids we investigated Collembola feeding preferences and types of ingested resources. We offered Collembola two resources with distinct isotope signature: a C4 plant (Zea mays L.) planted in soil with 15N labelled litter of Lolium perenne L. (C3 plant). We hypothesised that Collembola obtain their nutrients (C and N) from different resources, with their carbon being mainly derived from resources that are closely associated to the plant root e.g. root exudates causing enrichment in 13C in Collembola tissue, while the incorporated nitrogen originates from litter resources. In contrast to our hypothesis, bulk stable isotope analysis and compound specific analysis of fatty acids suggest that Collembola derived the majority of incorporated C and N from plant roots. Fatty acid δ13C and tissue δ13C and δ15N ratios of Collembola resembled those of maize plants. Furthermore of 13C signatures of fatty acids in Collembola corresponded to fatty acids in maize rather than to fatty acids from soil microorganisms. The results indicate that Collembola in the rhizosphere of plants, being assumed to be mainly decomposers, predominately live on plant resources, CHAPTER 4 44 presumably fine roots or root hairs i.e. are herbivorous rather than decomposers or fungivorous. 4.2 Introduction Collembola are among the most abundant soil arthropods, reaching particularly high densities in the rhizosphere of plants. Collembola affect plant growth and nutrition as well as plant performance in a variety of ways (Theenhaus et al. 1999, Endlweber and Scheu 2006). Changes in plant growth in presence of Collembola often are ascribed to increased nutrient mineralization and an associated increased nutrient uptake by plants (Bardgett and Cook 1998, Bardgett and Chan 1999, Lussenhop and BassiRad 1995). In fact, Collembola increase plant nutrient contents but the effect varies between plant species and functional groups (Scheu et al. 1999, Kreuzer et al. 2004, Partsch et al. 2006). Collembola mobilise nutrients by grazing on fungi and bacteria and by the formation of nutrient rich patches, i.e. by depositing faecal pellets (Teuben 1991, Lussenhop 1992, Jones 1998, Filser 2002). The formation of nutrient rich patches and increased nutrient mineralization therein likely induces root proliferation toward these “hotspots” (van Vuuren et al. 1996, Hodge et al. 1999, Hodge et al. 2000). In fact, Collembola affect root performance and induce the production of longer and thinner roots (Endlweber and Scheu 2006, 2007). However, the changes in root performance are not necessarily associated with an increase in tissue nutrient concentration or plant biomass. The mechanisms responsible for the changes in root morphology in presence of Collembola therefore remain little understood. In particular, it need to be resolved if Collembola feed on root tissue or accidentally damage roots when feeding on fungal hyphae and organic matter in the vicinity of roots. Root hairs may be CHAPTER 4 45 ingested or at least damaged by Collembola, and damaged plant roots may induce changes in plant metabolism resulting in altered root performance. Detailed knowledge on the feeding behaviour of Collembola in situ is needed to disentangle Collembola-plant interrelationships. Collembola are generalist feeders, ingesting a wide variety of resources. They probably obtain nutrients such as N and C from different resources with the ratio of incorporated nutrients depending on food quality (Scheu and Folger 2004). The analysis of stable isotope ratios in combination with fatty acid analysis offers the opportunity for investigating the contribution of different food resources to the diet of Collembola and therefore may allow uncovering trophic relationships between Collembola, rhizosphere fungi and plants. Stable isotope ratios of animal tissue, in particular 13C signatures, reflect the stable isotope ratio of the resources ingested (Peterson and Frey 1987, Post et al. 2002). By offering two resources with different stable isotope signatures the proportion of incorporated C and N derived from both resources can be determined, and this has been used to analyse dietary preferences of Collembola in the laboratory (Scheu and Folger 2002, Chamberlain et al. 2006). Food resources with different 13C/12C ratios can easily be obtained by using plant materials originating from C3 or C4 plants due to the discrimination of 13C by Rubisco in the photosynthetic pathway of C3 plants. Another recently introduced tool to analyse trophic links between Collembola and their food resources is the evaluation of the composition and 13C signature of fatty acids in the diet and the consumer (Ruess et al. 2005, Chamberlain et al. 2006). Phospholipid fatty acids (PLFAs) as components of cell membranes differ in particular between bacteria and fungi but also between bacterial phyla. In consumers neutral fatty acids (NLFAs), i.e. storage lipids, are linked to the animal’s diet. Fatty acids of the diet in part are incorporated into animal tissue without or with slight CHAPTER 4 46 modifications. The evaluation of NLFA patterns combined with compound specific 13C analysis of fatty acids provides a unique and powerful tool to trace food resources of Collembola (Ruess et al. 2004, Ruess et al. 2005). In the present study we evaluated the diet of Collembola by offering resources with distinct isotope signature. We established a laboratory system consisting of a C4 plant (Zea mays L.) planted in soil mixed with 15N labelled litter of Lolium perenne L. (C3 plant). We hypothesized that (i) the diet of Collembola is based in large on carbon resources entering the soil via roots, such as root exudates, therefore Collembola in our laboratory system with maize will be enriched in 13C, and (ii) a large fraction of the nitrogen in Collembola tissue originates from litter resources, i.e. Collembola obtain N and C for tissue formation from different dietary resources. 4.3 Materials and Methods The experiment was conducted in microcosms (diameter 10 cm, height 25 cm), sealed with a 45 µm mesh at the bottom to allow drainage. Microcosms were filled with 1 kg soil taken from an arable field (Jena, Thuringia, Germany). Prior to adding to the microcosms the soil was sieved (4 mm mesh) and frozen at -20°C for defaunation. The experiment was set up in a two factorial design with the factors Litter (with and without) and Plant (Maize; with and without). Litter (1.25 g dry weight) consisting of thoroughly mixed 250 mg labelled (δ15N = 11084) and 1000 mg unlabelled Lolium perenne L. shoots was added to half of the microcosms. The litter was reduced to small pieces (<0.5mm) and homogeneously mixed into the soil. Plant treatments received one Maize seed (Zea mays L) per microcosm. The treatments were replicated 10 times and watered every other day with 15 ml deionised water. The microcosms were incubated in a temperature controlled greenhouse equipped CHAPTER 4 47 with lamps for increasing radiation and setting day/night cycles (16 h light, 18°C) and arranged in a complete randomised design. After germination of the maize seeds each treatment received 100 individuals of the Collembola species Protaphorura fimata Gisin taken from laboratory cultures. Microcosms were harvested after 8 weeks. Plant roots were washed, weighed and divided into two subsamples. Half of the subsamples were frozen at -20°C until further analysis. The other half was dried at 60°C for three days. Soil samples were taken prior to washing the roots. Half of the soil samples were stored at -20°C the other half dried at 60°C for three days. During the washing procedure, Collembola floating on the water surface were collected and frozen at -20°C. Collembola taken from treatments with plants were divided into two subsamples which were dried at 60°C or stored at -20°C, respectively. All other Collembola were dried at 60°C for stable isotope analysis. The dried soil and plant materials as well as the litter samples were milled in a ball mill (Retsch, Haan, Germany). Collembola and subsamples of the milled materials were weighed into tin capsules for stable isotope analysis. Stable isotope ratios of the samples were analysed in a system consisting of an elemental analyser (NA 1500, Carlo Erba, Milan) coupled with a mass spectrometer (MAT 251, Finnigan; Reineking et al. 1993). Acetanilide (C8H9NO; Merck, Darmstadt) was used for internal calibration. The ratio between 13C and 12C was expressed relative to that in Pee Dee Belemnite (marine limestone). For 15N atmospheric nitrogen served as primary standard. Ratios [‰] were calculated according to the following formula: δX = (R sample – R standard)/(R standard) x 1000 (Peterson and Fry 1987), with X representing the heavier isotope (15N or 13C), and R the ratio between the heavy and the light isotope (15N/14N respectively 13C/12C). The proportion of N incorporated in Collembola tissue CHAPTER 4 48 derived from litter was calculated by a two-source mixing model (Newman Gearing 1991): F=(δ15NCollembola +litter - δ15Nlitter)/ ( δ15Nmaize - δ15Nlitter)*100 Three samples from each treatment were chosen for fatty acid analysis of Collembola, soil microorganisms and root material. Lipids of Collembola and of microorganisms in soil were extracted by shaking in a single phase extraction solvent consisting of chloroform, methanol and citrate buffer in ratios of 1.0:2.0:0.8 (Bligh and Dyer 1959). After addition of distilled water and CHCl3 and centrifugation the chloroform fraction of each sample was transferred to a silicic acid column. The lipids were eluted by consecutively adding chloroform (neutral lipids), acetone (glycolipids) and methanol (phospholipids). For soil microorganisms phospholipids were used for further analysis, whereas neutral lipids were used for analyses of the fatty acid composition of Collembola. Plant material and samples of Collembola and soil microorganisms were saponified and methylated (procedure given for the Sherlock Microbial Identification System; MIDI, Newark, USA). The samples were stored at -20°C until further analysis. Samples were analysed by gas chromatography (Clarus 500 GC, PerkinElmer Inc., Waltham, Massachusetts, USA). Compound specific δ13C analysis of fatty acids was conducted in a gas- chromatography-combustion-isotope-ratio-monitoring-mass spectrometer system (GC-C-IRM-MS) consisting of a gas chromatograph (6890 Series, Agilent Technology, USA) coupled via a Conflow III interface (ThermoFinnigan, Germany) to a MAT 252 mass spectrometer (ThermoFinnigan, Germany). A select FAME polar capillary column (50 m, 0.25 mm i.d., film thickness 0.25 mm) was used for the separation of the fatty acid methyl esters. CHAPTER 4 49 4.4.1 Statistical analysis: Stable isotope