Fungal-specific PCR primers developed for analysis of the ITS region of environmental DNA extracts

BACKGROUND: The Internal Transcribed Spacer (ITS) regions of fungal ribosomal DNA (rDNA) are highly variable sequences of great importance in distinguishing fungal species by PCR analysis. Previously published PCR primers available for amplifying these sequences from environmental samples provide varying degrees of success at discriminating against plant DNA while maintaining a broad range of compatibility. Typically, it has been necessary to use multiple primer sets to accommodate the range of fungi under study, potentially creating artificial distinctions for fungal sequences that amplify with more than one primer set. RESULTS: Numerous sequences for PCR primers were tested to develop PCR assays with a wide range of fungal compatibility and high discrimination from plant DNA. A nested set of 4 primers was developed that reflected these criteria and performed well amplifying ITS regions of fungal rDNA. Primers in the 5.8S sequence were also developed that would permit separate amplifications of ITS1 and ITS2. A range of basidiomycete fruiting bodies and ascomycete cultures were analyzed with the nested set of primers and Restriction Fragment Length Polymorphism (RFLP) fingerprinting to demonstrate the specificity of the assay. Single ectomycorrhizal root tips were similarly analyzed. These primers have also been successfully applied to Quantitative PCR (QPCR), Length Heterogeneity PCR (LH-PCR) and Terminal Restriction Fragment Length Polymorphism (T-RFLP) analyses of fungi. A set of wide-range plant-specific primers were developed at positions corresponding to one pair of the fungal primers. These were used to verify that the host plant DNA was not being amplified with the fungal primers. CONCLUSION: These plant primers have been successfully applied to PCR-RFLP analyses of forest plant tissues from above- and below-ground samples and work well at distinguishing a selection of plants to the species level. The complete set of primers was developed with an emphasis on discrimination between plant and fungal sequences and should be particularly useful for studies of fungi where samples also contain high levels of background plant DNA, such as verifying ectomycorrhizal morphotypes or characterizing phylosphere communities

Influence of adverse soil conditions on the formation and function of Arbuscular mycorrhizas

The majority of plants have mycorrhizal fungi associated with them. Mycorrhizal fungi are ecologically significant because they form relationships in and on the roots of a host plant in a symbiotic association. The host plant provides the fungus with soluble carbon sources, and the fungus provides the host plant with an increased capacity to absorb water and nutrients from the soil. Adverse conditions are a pervasive feature in both natural and agronomic soils. The soil environment is constantly changing with regard to moisture, temperature and nutrient availability. In addition, soil properties are often manipulated to improve crop yields. In many cases, soils may be contaminated through disposal of chemicals that are toxic to plants and microorganisms. The formation and function of mycorrhizal relationships are affected by edaphic conditions such as soil composition, moisture, temperature, pH, cation exchange capacity, and also by anthropogenic stressors including soil compaction, metals and pesticides. Arbuscular mycorrhizal fungi are of interest for their reported roles in alleviation of diverse soil-associated plant stressors, including those induced by metals and polychlorinated aliphatic and phenolic pollutants. Much mycorrhizal research has investigated the impact of extremes in water, temperature, pH and inorganic nutrient availability on mycorrhizal formation and nutrient acquisition. Evaluation of the efficacy of plant–mycorrhizal associations to remediate soils contaminated with toxic materials deserves increased attention. Before the full potential benefits of arbuscular mycorrhizal fungi to reclaim contaminated soils can be realized, research advances are needed to improve our understanding of the physiology of mycorrhizae subjected to adverse physical and chemical conditions. This paper will review literature and discuss the implications of soil contamination on formation and function of arbuscular mycorrhizal associations

Arbuscular mycorrhizal response to adverse soil conditions

Adverse conditions are a pervasive feature in both natural as well as agronomic soils. The soil environment is constantly changing with regard to moisture, temperature and nutrition. In addition, soil properties such as fertility, pH and aeration are often changed to improve crop yields. Soils have been unintentionally contaminated as a result of accidents that occur during agronomic operations or intentionally contaminated in mining or manufacturing operations by disposal of chemicals that are toxic to plants and micro-organisms. Mycorrhizal associations in terrestrial ecosystems influence organic and inorganic nutrient relationships, water relations and carbon cycling in plants. Relatively little is known about factors that control the vigour and extent of mycorrhization. This lack of understanding arises in large part from the difficulty of studying the intact association, which is a functionally and anatomically distinct structure comprising two biologically different organisms, e.g., plants and arbuscular mycorrhizae (AM) fungi. The formation and function of mycorrhizal relationships are affected by edaphic conditions such as soil composition, moisture, temperature, pH, cation exchange capacity. They are also affected by anthropogenic stressers such as heavy metals, pesticides and soil compaction. An organism's response to stress may involve interactions among various avoidance and tolerance mechanisms (Taylor, 1978; Tingey and Taylor, 1982; Tingey and Anderson, 1991). Stress avoidance mechanisms influence the amount and rate at which stress will reach the target site in the plant. Stress tolerance is defined as resistance via an ability "to come to thermodynamic equilibrium to the stress" without being killed (Levitt, 1980). In this chapter, we shall review the effects of a number of soil-associated stressers, including soil moisture, temperature, pH, heavy metals, agricultural practices and pesticides on AM development and function and host plant tolerance to these stresses. Several publications have reviewed the impact of various stresses on plant-mycorrhizal interactions (Anderson and Rygiewicz, 1991; Read, 1991; Van Duin et al, 1991; Sylvia and Williams, 1992), which provide additional information on this subject

Elevated CO2 and temperature impacts on different components of soil CO2 efflux in Douglas-fir terracosms

Although numerous studies indicate that increasing atmospheric CO2 or temperature data are available on the responses of three major components of soil respiration [i.e. rhizosphere respiration (root and root exudates), litter decomposition, and oxidation of soil organic matter] to different CO2 and temperature conditions. In this study, we applied a dual stable isotope approach to investigate the impact of elevated CO2 and elevated temperature on these components of soil CO2 efflux in Douglas-fir terracosms. We measured both soil CO2 efflux rates and the C-13 and O-18 isotopic compositions of soil CO2 efflux in 12 sun-lit and environmentally controlled terracosms with 4-year-old Douglas fir seedlings and reconstructed forest soils under two CO2 concentrations (ambient and 200 ppmv above ambient) and two air temperature regimes (ambient and 4 degrees C above ambient). The stable isotope data were used to estimate the relative contributions of different components to the overall soil CO2 efflux. In most cases, litter decomposition was the dominant component of soil CO2 efflux in this system, followed by rhizosphere respiration and soil organic matter oxidation. Both elevated atmospheric CO2 concentration and elevated temperature stimulated rhizosphere respiration and litter decomposition. The oxidation of soil organic matter was stimulated only by increasing temperature. Release of newly fixed carbon as root respiration was the most responsive to elevated CO2, while soil organic matter decomposition was most responsive to increasing temperature. Although some assumptions associated with this new method need to be further validated, application of this dual-isotope approach can provide new insights into the responses of soil carbon dynamics in forest ecosystems to future climate changes

Do mycorrhizal network benefits to survival and growth of interior Douglas-fir seedlings increase with soil moisture stress?

Facilitation of tree establishment by ectomycorrhizal (EM) networks (MNs) may become increasingly important as drought stress increases with climate change in some forested regions of North America. The objective of this study was to determine (1) whether temperature, CO2 concentration ([CO2]), soil moisture, and MNs interact to affect plant establishment success, such that MNs facilitate establishment when plants are the most water stressed, and (2) whether transfer of C and water between plants through MNs plays a role in this. We established interior Douglas-fir (Pseudotsuga menziesiivar.glauca) seedlings in root boxes with and without the potential to form MNs with nearby conspecific seedlings that had consistent access to water via their taproots. We varied temperature, [CO2], and soil moisture in growth chambers. Douglas-fir seedling survival increased when the potential existed to form an MN. Growth increased with MN potential under the driest soil conditions, but decreased with temperature at 800 ppm [CO2]. Transfer of 13C to receiver seedlings was unaffected by potential to form an MN with donor seedlings, but deuterated water (D2O) transfer increased with MN potential under ambient [CO2]. Chlorophyll fluorescence was reduced when seedlings had the potential to form an MN under high [CO2] and cool temperatures. We conclude that Douglas-fir seedling establishment in laboratory conditions is facilitated by MN potential where Douglas-fir seedlings have consistent access to water. Moreover, this facilitation appears to increase as water stress potential increases and water transfer via networks may play a role in this. These results suggest that conservation of MN potential may be important to forest regeneration where drought stress increases with climate change

Nitrate deposition in northern hardwood forests and the nitrogen metabolism of Acer saccharum marsh

It is generally assumed that plant assimilation constitutes the major sink for anthropogenic Nitrate NO 3 − deposited in temperate forests because plant growth is usually limited by nitrogen (N) availability. Nevertheless, plants are known to vary widely in their capacity for NO 3 − uptake and assimilation, and few studies have directly measured these parameters for overstory trees. Using a combination of field and greenhouse experiments, we studied the N nutrition of Acer saccharum Marsh. in four northern hardwood forests receiving experimental NO 3 − additions equivalent to 30 kg N ha −1 year −1 . We measured leaf and fine-root nitrate reductase activity (NRA) of overstory trees using an in vivo assay and used 15 N to determine the kinetic parameters of NO 3 − uptake by excised fine roots. In two greenhouse experiments, we measured leaf and root NRA in A. saccharum seedlings fertilized with 0–3.5 g NO 3 − −N m −2 and determined the kinetic parameters of NO 3 − and NH 4 + uptake in excised roots of seedlings. In both overstory trees and seedlings, rates of leaf and fine root NRA were substantially lower than previously reported rates for most woody plants and showed no response to NO 3 − fertilization (range = non-detectable to 33 nmol NO 2 − g −1 h −1 ). Maximal rates of NO 3 − uptake in overstory trees also were low, ranging from 0.2 to 1.0 μmol g −1 h −1 . In seedlings, the mean V max for NO 3 − uptake in fine roots (1 μmol g −1 h −1 ) was approximately 30 times lower than the V max for NH 4 + uptake (33 μmol g −1 h −1 ). Our results suggest that A. saccharum satisfies its N demand through rapid NH 4 + uptake and may have a limited capacity to serve as a direct sink for atmospheric additions of NO 3 − .Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/47695/1/442_2004_Article_BF00334659.pd

An Adaptive Learning System Using Correlation and Steepest Descent Optimization Techniques

This thesis is concerned with the identification of multiparameter systems by the use of adaptive multiparameter learning models. Multiparameter adjustment signals based on the differences between the corresponding model and system parameters are obtained by cross-correlating two signals within the adaptive system. The adjustment signals are fed back to the model to adjust its parameters such that the Mean Square Error is minimized along its path of steepest descent in parameter space. The Mean Square Error is a minimum when the corresponding system and model parameters are matched and thus indicates identification of the system by the model. The results obtained for the multiparameter case are applied to the identification of the parameters corresponding to the damping ratio and natural frequency of a second order system by adjustment of the corresponding parameters of a second order model. Analog simulation studies of the second order example are conducted in order to demonstrate and validate the theory

Information systems safety in a context of autmotive applications

Strong expansion of IT tools in area of management and control of systems and devices of road transport is connected inseparably with a need of assurance of proper level of credibility and data safety. In paper one related to issues connected with threatens and ways of their reduction in a context of selected law regulation and analysis obtained during studies on modern motion sensor applied in system of digital tachograph in vehicles from delivery trucks and buses group. Departures from assumed scheme of information flow in vehicle can result from random interfering environmental factors as well as , action taken deliberately for specific purpose, as change of object's operation (e.g. tuning of power transmission system), or illicit modification of data subjected to revision by institutions obligated to control of vehicle. Presented in the paper example of threads and methods of information protection are part of the whole system of defined threats and securities necessary to obtain applied certificates granted by selected institutions such as BSI (Bundesamt für Sicherheit in der Informationstechnik) and development of so called PPprotection profile. In summary there are also pointed another ways to enhance a security level of described system such as e.g. redundancy in acquiring and transmission of data