Extraction methods in soil phosphorus characterisation : Limitations and applications

The quantification and characterisation of soil phosphorus (P) is of agricultural and environmental importance and different extraction methods are widely used to asses the bioavailability of P and to characterize soil P reserves. However, the large variety of extractants, pre-treatments and sample preparation procedures complicate the comparison of published results. In order to improve our understanding of the behaviour and cycling of P in soil, it is crucial to know the scientific relevance of the methods used for various purposes. The knowledge of the factors affecting the analytical outcome is a prerequisite for justified interpretation of the results. The aim of this thesis was to study the effects of sample preparation procedures on soil P and to determine the dependence of the recovered P pool on the chemical nature of extractants. Sampling is a critical step in soil testing and sampling strategy is dependent on the land-use history and the purpose of sampling. This study revealed that pre-treatments changed soil properties and air-drying was found to affect soil P, particularly extractable organic P, by disrupting organic matter. This was evidenced by an increase in the water-extractable small-sized (<0.2 µm) P that, at least partly, took place at the expense of the large-sized (>0.2 µm) P. However, freezing induced only insignificant changes and thus, freezing can be taken to be a suitable method for storing soils from the boreal zone that naturally undergo periodic freezing. The results demonstrated that chemical nature of the extractant affects its sensitivity to detect changes in soil P solubility. Buffered extractants obscured the alterations in P solubility induced by pH changes; however, water extraction, though sensitive to physicochemical changes, can be used to reveal short term changes in soil P solubility. As for the organic P, the analysis was found to be sensitive to the sample preparation procedures: filtering may leave a large proportion of extractable organic P undetected, whereas the outcome of centrifugation was found to be affected by the ionic strength of the extractant. Widely used sequential fractionation procedures proved to be able to detect land-use -derived differences in the distribution of P among fractions of different solubilities. However, interpretation of the results from extraction experiments requires better understanding of the biogeochemical function of the recovered P fraction in the P cycle in differently managed soils under dissimilar climatic conditions.

Pathogen-Induced Defense Signaling and Signal Crosstalk in Arabidopsis

Erwinia carotovora subsp. carotovora is a bacterial phytopathogen that causes soft rot in various agronomically important crop plants. A genetically specified resistance to E. carotovora has not been defined, and plant resistance to this pathogen is established through nonspecific activation of basal defense responses. This, together with the broad host range, makes this pathogen a good model for studying the activation of plant defenses. Production and secretion of plant cell wall-degrading enzymes (PCWDE) are central to the virulence of E. carotovora. It also possesses the type III secretion system (TTSS) utilized by many Gram-negative bacteria to secrete virulence- promoting effector proteins to plant cells. This study elucidated the role of E. carotovora HrpN (HrpNEcc), an effector protein secreted through TTSS, and the contribution of this protein in the virulence of E. carotovora. Treatment of plants with HrpNEcc was demonstrated to induce a hypersensitive response (HR) as well as resistance to E. carotovora. Resistance induced by HrpNEcc required both salicylic acid (SA)- and jasmonate/ethylene (JA/ET)-dependent defense signaling in Arabidopsis. Simultaneous treatment of Arabidopsis with HrpNEcc and PCWDE polygalacturonase PehA elicited accelerated and enhanced induction of defense genes but also increased production of superoxide and lesion formation. This demonstrates mutual amplification of defense signaling by these two virulence factors of E. carotovora. Identification of genes that are rapidly induced in response to a pathogen can provide novel information about the early events occurring in the plant defense response. CHLOROPHYLLASE 1 (AtCLH1) and EARLY RESPONSIVE TO DEHYDRATION 15 (ERD15) are both rapidly triggered by E. carotovora in Arabidopsis. Characterization of AtCLH1 encoding chlorophyll-degrading enzyme chlorophyllase indicated that it might have a role in chlorophyll degradation during plant tissue damage. Silencing of this gene resulted in increased accumulation of reactive oxygen species (ROS) in response to pathogen infection in a light-dependent manner. This led to enhanced SA-dependent defenses and resistance to E. carotovora. Moreover, crosstalk between different defense signaling pathways was observed; JA-dependent defenses and resistance to fungal pathogen Alternaria brassicicola were impaired, indicating antagonism between SA- and JA-dependent signaling. Characterization of ERD15 suggested that it is a novel, negative regulator of abscisic acid (ABA) signaling in Arabidopsis. Overexpression of ERD15 resulted in insensitivity to ABA and reduced tolerance of the plants to dehydration stress. However, simultaneously, the resistance of the plants to E. carotovora was enhanced. Silencing of ERD15 improved freezing and drought tolerance of transgenic plants. This, together with the reducing effect of ABA on seed germination, indicated hypersensitivity to this phytohormone. ERD15 was hypothesized to act as a capacitor that controls the appropriate activation of ABA responses in Arabidopsis.

Behavioural and physiological responses to predators of captive-bred Arctic charr: significance of genetics, learning and ontogeny

Predation forms one of the main selective forces in nature and in a vast number of prey species the behavioural responses form the main way to avoid predation. World wide numerous captive breeding programs are used to produce fish and other animal species for conservational reintroductions. However, rearing animals in the absence of predators in captivity has been shown to weaken their predator avoidance skills and lead to behavioural divergence between wild and captive-bred populations. In my thesis I studied the effects of predator odour exposures on antipredator behavioural and physiological responses of captive reared Saimaa Arctic charr. This charr population is the most endangered fish population in Finland and a sample of the remaining population has been taken to captive breeding and used for an extensive reintroduction program. Lowered responsiveness to predators is probably one of the major reasons for the poor survival probability of the charr after release into the wild. The main aims of my thesis were to explore the reasons for behavioural phenotypic variation in this charr population and whether naïve charr young could be trained to recognise their natural predators. The predator species in my thesis were burbot (Lota lota) and pikeperch (Sander lucioperca). In my thesis I showed that the captive-bred charr responded to chemical cues from burbot and pikeperch, but the magnitude of responses was linked to the predator species. The burbot odour increased the spatial odour avoidance of the charr young. On the other hand, in the pikeperch treatment charr reduced their relative swimming activity and tended to show more freezing behaviour relative to the burbot treatment. It seems evident that these different responses are related to the different hunting tactics of predator species. Furthermore, I detected wide between-family differences in antipredator responsiveness (i.e. inherited variation in antipredator behaviours) in this captive stock. Detected differences were greater in the response towards burbot than towards pikeperch. These results, in addition to predator-specific antipredator responses, suggest that there is a clear inherited component in antipredator responsiveness in Saimaa charr population and that the detected inherited differences could explain a part of the behavioural phenotypic variation in this population. In my thesis I also found out that both social learning and direct exposure to live predators enhance the antipredator responsiveness of charr young. In addition, I obtained indications that predator odour exposures (i.e. life-skills training) in alevin and fry stages can fine-tune the innate antipredator responsiveness of charr. Thus, all these methods have the potential to enhance the innate antipredator responsiveness of naïve charr young, possibly also improving the post-release survival of these trained individuals in the wild. However, the next logical phase would be to carry out large scale survival studies in the wild to test this hypothesis. Finally, the results of my thesis emphasize that possible long-term life-skills training methods should take into account not only the behavioural but also the physiological effects of training.

New approaches to improve the cord blood unit hematopoietic progenitor cell content

Cord blood is a well-established alternative to bone marrow and peripheral blood stem cell transplantation. To this day, over 400 000 unrelated donor cord blood units have been stored in cord blood banks worldwide. To enable successful cord blood transplantation, recent efforts have been focused on finding ways to increase the hematopoietic progenitor cell content of cord blood units. In this study, factors that may improve the selection and quality of cord blood collections for banking were identified. In 167 consecutive cord blood units collected from healthy full-term neonates and processed at a national cord blood bank, mean platelet volume (MPV) correlated with the numbers of cord blood unit hematopoietic progenitors (CD34+ cells and colony-forming units); this is a novel finding. Mean platelet volume can be thought to represent general hematopoietic activity, as newly formed platelets have been reported to be large. Stress during delivery is hypothesized to lead to the mobilization of hematopoietic progenitor cells through cytokine stimulation. Accordingly, low-normal umbilical arterial pH, thought to be associated with perinatal stress, correlated with high cord blood unit CD34+ cell and colony-forming unit numbers. The associations were closer in vaginal deliveries than in Cesarean sections. Vaginal delivery entails specific physiological changes, which may also affect the hematopoietic system. Thus, different factors may predict cord blood hematopoietic progenitor cell numbers in the two modes of delivery. Theoretical models were created to enable the use of platelet characteristics (mean platelet volume) and perinatal factors (umbilical arterial pH and placental weight) in the selection of cord blood collections with high hematopoietic progenitor cell counts. These observations could thus be implemented as a part of the evaluation of cord blood collections for banking. The quality of cord blood units has been the focus of several recent studies. However, hemostasis activation during cord blood collection is scarcely evaluated in cord blood banks. In this study, hemostasis activation was assessed with prothrombin activation fragment 1+2 (F1+2), a direct indicator of thrombin generation, and platelet factor 4 (PF4), indicating platelet activation. Altogether three sample series were collected during the set-up of the cord blood bank as well as after changes in personnel and collection equipment. The activation decreased from the first to the subsequent series, which were collected with the bank fully in operation and following international standards, and was at a level similar to that previously reported for healthy neonates. As hemostasis activation may have unwanted effects on cord blood cell contents, it should be minimized. The assessment of hemostasis activation could be implemented as a part of process control in cord blood banks. Culture assays provide information about the hematopoietic potential of the cord blood unit. In processed cord blood units prior to freezing, megakaryocytic colony growth was evaluated in semisolid cultures with a novel scoring system. Three investigators analyzed the colony assays, and the scores were highly concordant. With such scoring systems, the growth potential of various cord blood cell lineages can be assessed. In addition, erythroid cells were observed in liquid cultures of cryostored and thawed, unseparated cord blood units without exogenous erythropoietin. This was hypothesized to be due to the erythropoietic effect of thrombopoietin, endogenous erythropoietin production, and diverse cell-cell interactions in the culture. This observation underscores the complex interactions of cytokines and supporting cells in the heterogeneous cell population of the thawed cord blood unit.

Overwintering ecology of northern field layer plants : snow and photosynthesis in Vaccinium vitis-idaea L.

Winter is a significant period for the seasonality of northern plants, but is often overlooked when studying the interactions of plants and their environment. This study focuses on the effects of overwintering conditions, including warm winter periods, snow, and snowmelt on boreal and sub-Arctic field layer plants. Wintertime photosynthesis and related physiological factors of evergreen dwarf shrubs, particularly of Vaccinium vitis-idaea, are emphasised. The work combines experiments both in the field and in growth chambers with measurements in natural field conditions. Evergreen dwarf shrubs are predominantly covered by snow in the winter. The protective snow cover provides favourable conditions for photosynthesis, especially during the spring before snowmelt. The results of this study indicate that photosynthesis occurs under the snow in V. vitis-idaea. The light response of photosynthesis determined in field conditions during the period of snow cover shows that positive net CO2 exchange is possible under the snow in the prevailing light and temperature. Photosynthetic capacity increases readily during warm periods in winter and the plants are thus able to replenish carbohydrate reserves lost through respiration. Exposure to low temperatures in combination with high light following early snowmelt can set back photosynthesis as sustained photoprotective measures are activated and photodamage begins to build up. Freezing may further decrease the photosynthetic capacity. The small-scale distribution of many field layer plants, including V. vitis-idaea and other dwarf shrubs, correlates with the snow distribution in a forest. The results of this study indicate that there are species-specific differences in the snow depth affinity of the field and ground layer species. Events and processes taking place in winter can have a profound effect on the overall performance of plants and on the interactions between plants and their environment. Understanding the processes involved in the overwintering of plants is increasingly important as the wintertime climate in the north is predicted to change in the future.

Microbiological characterisation of soils : Evaluation of some critical steps in data collection and experimental design

The study of soil microbiota and their activities is central to the understanding of many ecosystem processes such as decomposition and nutrient cycling. The collection of microbiological data from soils generally involves several sequential steps of sampling, pretreatment and laboratory measurements. The reliability of results is dependent on reliable methods in every step. The aim of this thesis was to critically evaluate some central methods and procedures used in soil microbiological studies in order to increase our understanding of the factors that affect the measurement results and to provide guidance and new approaches for the design of experiments. The thesis focuses on four major themes: 1) soil microbiological heterogeneity and sampling, 2) storage of soil samples, 3) DNA extraction from soil, and 4) quantification of specific microbial groups by the most-probable-number (MPN) procedure. Soil heterogeneity and sampling are discussed as a single theme because knowledge on spatial (horizontal and vertical) and temporal variation is crucial when designing sampling procedures. Comparison of adjacent forest, meadow and cropped field plots showed that land use has a strong impact on the degree of horizontal variation of soil enzyme activities and bacterial community structure. However, regardless of the land use, the variation of microbiological characteristics appeared not to have predictable spatial structure at 0.5-10 m. Temporal and soil depth-related patterns were studied in relation to plant growth in cropped soil. The results showed that most enzyme activities and microbial biomass have a clear decreasing trend in the top 40 cm soil profile and a temporal pattern during the growing season. A new procedure for sampling of soil microbiological characteristics based on stratified sampling and pre-characterisation of samples was developed. A practical example demonstrated the potential of the new procedure to reduce the analysis efforts involved in laborious microbiological measurements without loss of precision. The investigation of storage of soil samples revealed that freezing (-20 °C) of small sample aliquots retains the activity of hydrolytic enzymes and the structure of the bacterial community in different soil matrices relatively well whereas air-drying cannot be recommended as a storage method for soil microbiological properties due to large reductions in activity. Freezing below -70 °C was the preferred method of storage for samples with high organic matter content. Comparison of different direct DNA extraction methods showed that the cell lysis treatment has a strong impact on the molecular size of DNA obtained and on the bacterial community structure detected. An improved MPN method for the enumeration of soil naphthalene degraders was introduced as an alternative to more complex MPN protocols or the DNA-based quantification approach. The main advantage of the new method is the simple protocol and the possibility to analyse a large number of samples and replicates simultaneously.