Field application of a DNA-based assay to the measurement of roots of perennial grasses

Background and aims

DNA-based methods present new opportunities for overcoming the difficulties of accurately identifying and quantifying roots of different plant species in field soils. In order to quantify species-specific root biomass from measurements of DNA, consideration needs to be given to replication and ability to recover roots for calibration purposes in order to account for spatial, temporal and inter- and intra-species variation in DNA content of roots and distribution of roots within the soil profile.

Methods

This paper develops the field application of a DNA-based technique for direct quantification of roots in soils. The method was applied to a field experiment to investigate differences in root growth of acid-soil resistant and sensitive genotypes of perennial pasture grasses in an acid soil. DNA was extracted directly from soil and species-specific DNA was quantified using quantitative real-time PCR prior to estimation of root biomass.

Results

Root growth of the perennial grasses was quantified using the DNA-based technique, although separate calibration procedures were needed to convert DNA content to root mass for each species, soil layer and sampling date. Compared to acid-soil resistant genotypes, lesser root growth in acid soil layers and reduced above-ground dry matter production was observed for acid-soil sensitive genotypes.

Conclusions

The DNA-based method allowed genotypic differences in root growth to be assessed directly in soil and was advantageous for rapid processing of a large number of samples. However, high replication was still required to overcome spatial variability and separate calibrations were required for different species and soil depths across sampling times. The technique demonstrated greater root growth of acid-soil resistant perennial grasses which was beneficial for their establishment and persistence.     

Effect of soil acidity, soil strength and macropores on root growth and morphology of perennial grass species differing in acid-soil resistance

It is unclear whether roots of acid-soil resistant plants have significant advantages, compared with acid-soil sensitive genotypes, when growing in high-strength, acid soils or in acid soils where macropores may allow the effects of soil acidity and strength to be avoided. The responses of root growth and morphology to soil acidity, soil strength and macropores by seedlings of five perennial grass genotypes differing in acid-soil resistance were determined, and the interaction of soil acidity and strength for growth and morphology of roots was investigated. Soil acidity and strength altered root length and architecture, root hair development, and deformed the root tip, especially in acid-soil sensitive genotypes. Root length was restricted to some extent by soil acidity in all genotypes, but the adverse impact of soil acidity on root growth by acid-soil resistant genotypes was greater at high levels of soil strength. Roots reacted to soil acidity when growing in macropores, but elongation through high-strength soil was improved. Soil strength can confound the effect of acidity on root growth, with the sensitivity of acid-resistant genotypes being greater in high-strength soils. This highlights the need to select for genotypes that resist both acidity and high soil strength.     

Quantification of roots and seeds in soil with real-time PCR

Study of roots and associated organisms in soil particularly in mixed plant populations, such as pastures, is limited by difficulties in quantification of root growth and function. The research evaluated the potential of DNA quantification by real-time PCR to improve our capacity to study and understand roots in such contexts. Probes and primers were developed for two common pasture species, Trifolium subterraneum and Lolium perenne (and closely related Lolium spp.), and evaluated for specificity and sensitivity in TaqMan assays on DNA extracted from soil. Further experiments examined the ability to detect DNA in dead roots, the changes in root DNA levels of plants defoliated or treated with herbicide and the relationship between DNA and root dry weight for single and mixed plant species grown in pots. T. subterraneum DNA/PCR 200 fg/µl was detected at 17.5 cycles and L. perenne at 19.5 cycles. The assay for T. subterraneum was species specific but the L. perenne assay, as anticipated from the choice of probe, also detected some closely related species. The assays were sensitive and capable of detecting equivalent to <2 mg roots/kg of dry soil and able to quantify targets in mixed populations. DNA concentration varied with plant age and genotype and DNA in dead roots found to decay rapidly over a few days. DNA concentrations in roots were found to respond more rapidly to defoliation and herbicide treatments than root mass. This approach appears to offer a new way to study roots in soil and indicates that quantifying root DNA could provide insights into root function and responses not readily provided by other methods.     

Investigations into the exploitation of heterogeneous soils by Lupinus albus L. and L. pilosus Murr. and the effect upon plant growth

In calcareous soils, genotypes of Lupinus albus L. generally grow poorly, resulting in stunted plants that often develop lime-induced chlorosis. In contrast, some genotypes of L. pilosus Murr. occur naturally in calcareous soils without developing any visible symptoms of stress. Some genotypic variation for tolerance to calcareous soil does exist in L. albus and the tolerance mechanisms need to be determined. The adaptation through root system morphological plasticity of L. albus and L. pilosus, to heterogeneous limed soil profiles (pH 7.8) containing either patches of acid (non-limed) soil, or vertically split between acid and limed soil, was investigated. When grown in the presence of patches of acid soil, L. albus had a 52% greater shoot dry weight and visibly greener leaves compared with plants grown in the homogeneous limed soil. Total root dry matter in the acid-soil patches was greater than in the control limed-soil patches. This was due to a four-fold increase in the cluster root mass, accounting for 95% of the root dry matter in the acid-soil patch. Although these cluster roots secreted no more citric acid per unit mass than those in the limed soil did, their greater mass resulted in a higher citrate concentration in the surrounding soil. L. pilosus responded to the patches of acid soil in a manner comparable with L. albus. When grown in the homogeneous limed soil, L. pilosus had a greater maximum net CO₂ assimilation rate (P_max) than L. albus, however, the P_max of both species increased after they had accessed a patch of acid soil. Differences were apparent between the L. albus genotypes grown in soil profiles split vertically into limed and acid soil. A genotype by soil interaction occurred in the partitioning between soils of the cluster roots. The genotype La 674 was comparable with L. pilosus and produced over 11% of its cluster roots in the limed soil, whereas the other genotypes produced only 1–3% of their cluster roots in the limed soil. These results indicate L. pilosus is better adapted to the limed soil than L. albus, but that both species respond to a heterogeneous soil by producing mainly cluster roots in an acid-soil patch. This revised version was published online in June 2006 with corrections to the Cover Date.     

The relationship between silicon availability,and growth and silicon concentration of the salt marsh halophyte Spartina anglica

Incorporation of cover crops into cropping systems may contribute to a more efficient utilization of soil and fertilizer P by less P-efficient crops through exudation of P-mobilizing compounds by the roots of P-efficient plant species. The main objective of the present work was to test this hypothesis. First a method has been developed which allows the quantification of organic anion exudation from individual cluster roots formed by P-deficient white lupin (Lupinus albus L.). Lupin plants were grown in nutrient solution at 1 μM P and in a low P loess in small rhizotrons. Organic anions exuded from intact plants grown in nutrient solution were collected from individual cluster roots and root tips sealed in small compartments by an anion-exchange resin placed in nylon bags (resin-bags). Succinate was the dominant organic anion exuded followed by citrate and malate. The mean of citrate exudation-rate was 0.06 pmol mm⁻¹ s⁻¹ with exudation highly dependent on the citrate concentration and on the age of the cluster roots. Exudates from cluster roots and root tips grown at the soil surface (rhizotron-grown plants) were collected using overlayered resin–agar (resin mixed with agar). Citrate exudation from cluster roots was 10 times higher than that from root tips. Fractionation of P in the cluster root rhizosphere-soil indicates that white lupin can mobilize P not only from the available and acid-soluble P, but also from the stable residual soil P fractions. In pot experiments with an acid luvisol derived from loess low in available P, growth of wheat was significantly improved when mixed-cropped with white lupin due to improved P uptake. Both in mixed culture and in rotation wheat could benefit from the P mobilization capacity of white lupin, supporting the hypothesis above. Nine tropical leguminous cover crops and maize were grown in a pot experiment using a luvisol from Northern Nigeria low in available P. All plant species derived most of their P from the resin and bicarbonate-extractable inorganic P. Organic P (P_o) accumulated particularly in the rhizosphere of all plant species. There was a significant negative correlation between the species-specific rhizosphere acid phosphatase activity and P_o accumulation. Growth and P uptake of maize grown in rotation after legumes were enhanced indicating that improved P nutrition was a contributing factor. This revised version was published online in June 2006 with corrections to the Cover Date.     

Interspecific differences in root architecture among maize and triticale genotypes grown under drought,waterlogging and soil compaction

Environmental stresses (soil compaction, drought, waterlogging) cause changes in plants’ root system structure, also affecting the growth of above-ground parts. The aim of this study was to estimate phenotypic variation among maize and triticale genotypes in root penetration ability through petrolatum-wax-layer (RPA). Also, the effect of shortage or excess of soil water on dry matter of shoots and roots and morphological changes in root system structure in sensitive and resistant maize and triticale genotypes grown in low or high soil compaction level was evaluated. To estimate RPA index, the petrolatum-wax-layer method (PWL) was used. The strength of three petrolatum-wax concentrations 60, 50 and 40 % was 0.52, 1.07 and 1.58 MPa, respectively. High coefficients of variation (CV) were observed in 0.52 and 1.07 MPa and for maize were 19.2 and 21.7 %, and for triticale, 12.5 and 18.3 %, respectively. The data indicate that the use of PWL technique is an effective screening method, and makes it possible to divide the genotypes into resistant and sensitive groups. The second part of this study investigated a multistress effect of soil compaction combined with drought or waterlogging on root and shoot growth and morphological changes in root system structure of maize and triticale genotypes differing in susceptibility to environmental stresses. Seedlings were grown for 4 weeks in root-boxes under conditions of low (LSC 1.1 g cm^?3) or severe (SSC 1.6 g cm^?3) soil compaction. Drought or waterlogging stresses were applied for 2 weeks from 14th to 28th day. In comparison to LSC treatment, in SSC treatment the decrease in dry matter of shoots and roots was greater for sensitive genotypes of maize and triticale (Ancora, CHD-147). Soil drought or waterlogging caused greater decrease of dry matter of shoots and roots in seedlings grown in SSC in comparison to LSC. The root penetration index (RPI) was estimated as a ratio of root dry matter in 15–40 cm root-box layer to total root dry matter. On the basis of RPI it was possible to group the genotypes according to their ability to distribute roots in soil profile. In comparison to LSC, SSC exerted a strong influence on the length of seminal and seminal adventitious roots, as well as the number and length of L- and S-type lateral roots developed on seminal and nodal roots. In both species the restriction effect of soil compaction on number and length of roots was more severe in sensitive (Ankora, CHD-147) than in resistant (Tina, CHD-247) genotypes. The restriction in roots propagation was greater in triticale than in maize. Exposure to drought or waterlogging in the case of genotypes grown in LSC and SSC treatments caused a decrease in number and length of particular components of root system structure. In both species the decrease of root number and length in plants grown under waterlogging was greater than under drought. The observed changes in root system were greater in sensitive (Ankora, CHD147) than in resistant (Tina, CHD-247) genotypes. Statistically significant correlations were found between RPA and RPI and also between these indexes and soil compaction, drought and waterlogging susceptibility indexes. This indicates that genotypes resistant to soil compaction were resistant to drought or waterlogging and also that genotypes resistant to drought were resistant to waterlogging.     

Yield,root morphology and chemical composition of two pasture legumes as affected by lime and phosphorus applications to an acid soil

Summary Effects of increasing rates of lime (0, 900, 1725, and 3000 kg Ca(OH)₂/ha producing soil pH of 4.0, 4.7, 5.1 and 5.6) and P (50, 150, 250 and 350 kg P/ha) on top and root yield, root morphology and chemical composition of lotus (Lotus pedunculatus Cav.) and white clover (Trifolium repens L.), were studied, using an acid soil in a greenhouse experiment. Increasing rates of applied lime and phosphate resulted in substantial increases in top yields of both species but concomitant increases in root yield were small. In the unlimed soil, lotus out-yielded (tops and roots) white clover at all P levels. However, in the three limed treatments, white clover clearly out-yielded lotus. Yield response curves to applied P levelled off at the two highest lime rates for lotus but not for white clover. Nodulation and N content of white clover increased significantly with increasing lime applications, but for lotus there was a significant decrease in nodulation at the highest lime rate. Increased P rates had a small stimulatory effect on nodulation in both species. Of the total root weight, the percentage contribution of the tap and primary lateral root fractions was smaller and that of the secondary plus tertiary lateral roots was greater for lotus than for white clover although root length per unit weight tended to be larger for white clover at the two highest lime rates. Furthermore, lotus possessed longer and more numerous root hairs than white clover. Lime applications significantly decreased the percentage contribution of the tap and primary lateral roots to the total root weight and increased the percentage contribution of the secondary plus tertiary lateral roots. Al and Mn contents of tops and roots of both species decreased with increasing lime rates. There was a highly significant negative correlation between relative yield and Al content of lotus and white clover tops. In comparison with the limed treatments, in the unlimed treatments a greater percentage of total P, Al, Mn and N content accumulated in the roots of both species. In addition, lotus accumulated a much greater percentage Al in its roots than white clover.     

Root taxa identification in plant mixtures – current techniques and future challenges

Background

Studying root biomass, root system distribution and belowground interactions is essential for understanding the composition of plant communities, the impact of global change, and terrestrial biogeochemistry. Most soil samples and minirhizotron pictures hold roots of more than one species or plant individual. The identification of taxa by their roots would allow species-specific questions to be posed; information about root affiliation to plant individuals could be used to determine intra-specific competition.

Scope

Researchers need to be able to discern plant taxa by roots as well as to quantify abundances in mixed root samples. However, roots show less distinctive features that permit identification than aboveground organs. This review discusses the primary use of available methods, outlining applications, shortcomings and future developments.

Conclusion

Methods are either non-destructive, e.g. visual examination of root morphological criteria in situ, or require excavated and excised root samples. Among the destructive methods are anatomical keys, chemotaxonomic approaches and molecular markers. While some methods allow for discerning the root systems of individual plants, others can distinguish roots on the functional group or plant taxa level; methods such as IR spectroscopy and qPCR allow for quantifying the root biomass proportion of species without manual sorting.     

Simultaneous monitoring of two fungal genotypes on plant roots by single nucleotide polymorphism quantification with an innovative KASPar quantitative PCR

An innovative quantitative PCR-based method derived from the Kompetitive Allele Specific PCR Assay Reagent (KASPar) system was developed to quantify the genomic DNA from two coexisting genotypes on the same tissues of a host-plant. For this purpose, the classical end-point KASPar method was evolved to a real-time method thanks to the addition of an adapted measurement step after each PCR cycle. It was applied to the quantification of the two genotypes G1 and G2 of the Gaeumannomyces graminis var. tritici (Ggt) soilborne fungus, pathogenic on wheat roots. Specific primers targeting a single nucleotide polymorphism from the ITS sequence were used allowing simultaneous quantification of both genotypes in the same reaction. The assays were applied to quantify fungal DNA of each genotype, aside or mixed together, after DNA extraction from fungal pure cultures and from single or co-inoculated roots in artificial medium or in soil. The detection and quantification lower limits for the two genotypes were 1.25 pg and 5 pg for DNA from fungal pure cultures, and 1.8 pg and 7 pg for DNA from fungal inoculated roots. The advantages of this cost-effective method are the high levels of specificity, sensitivity and reproducibility. Moreover, the accuracy of the method is independent of the copy numbers of the target sequences. The method is the first one to adapt the non-quantitative genotyping KASPar system to a quantitative application of two known genotypes of a species simultaneously and is suitable for simultaneous genotype-specific quantification of any other organisms (fungi, bacteria, plants).     

Species interactions at the level of fine roots in the field: influence of soil nutrient heterogeneity and plant size

Interference at the level of fine roots in the field was studied by detailed examination of fine root distribution in small soil patches. To capture roots as they occur in natural three-dimensional soil space, we used a freezing and slicing technique for microscale root mapping. The location of individual roots intersecting a sliced soil core surface was digitized and the identity of shrub and grass roots was established by a chemical technique. Soil patches were created midway between the shrub, Artemisia tridentata, and one of two tussock grasses, Pseudoroegneria spicata or Agropyron desertorum. Some soil patches were enriched with nutrients and others given only deionized water (control); in addition, patches were located between plants of different size combination (large shrubs with small tussock grasses and small shrubs with large tussock grasses). The abundance of shrub and grass roots sharing soil patches and the inter-root distances of individual fine roots were measured. Total average rooting density in patches varied among these different treatment combinations by only a factor of 2, but the proportion of shrub and grass roots in the patches varied sixfold. For the shrub, the species of grass roots sharing the patches had a pronounced influence on shrub root density; shrub roots were more abundant if the patch was shared with Pseudoroegneria roots than if shared with Agropyron roots. The relative size of plants whose roots shared the soil patches also influenced the proportion of shrub and grass roots; larger plants were able to place more roots in the patches than were the smaller plants. In the nutrient-enriched patches, these influences of grass species and size combination were amplified. At the millimeter- to centimeter-scale within patches, shrub and grass roots tended to segregate, i.e., avoid each other, based on nearest-neighbor distances. At this scale, there was no indication that the species-specific interactions were the result of resource competition, since there were no obvious patterns between the proportion of shrub and grass roots of the two species combinations with microsite nutrient concentrations. Other potential mechanisms are discussed. Interference at the fine-root level, and its species-specific character, is likely an influential component of competitive success, but one that is not easily assessed.     

Nutrient uptake estimates for woody species as described by the NST 3.0, SSAND, and PCATS mechanistic nutrient uptake models

The effect of soil acidity on root and rhizosheath development in wheat and barley seedlings was investigated in an acid Ferrosol soil to which various amounts of lime (CaCO₃) were applied to modify soil Al concentrations (pH (CaCl₂): 4.22 to 5.35 and Al (CaCl₂ extract): 17.7 to 0.4 mg kg^?1 soil; respectively), and Ferrosol soil from an adjacent location at the same site which had a higher Al concentration (pH 4.19; 29.2 mg kg^?1 Al). The cereal lines were selected on the basis of differences in their rate of root growth, Al-resistance and root hair morphology. Root morphology was assessed after 7 days of growth. The length of fine (mainly lateral) roots of Al-sensitive genotypes was more sensitive to soil Al concentrations than that of the coarse (mainly primary) roots. The experiments demonstrated that even where root growth was protected by expression of the TaALMT1 gene for Al-resistance, root-soil contact was diminished by soil acidity because root hair length (in many lines), and root hair density and rhizosheath formation (all lines) were adversely affected by soil acidity. In the case of Al-sensitive lines, fine root growth and rhizosheath mass were reduced over much the same range of soil Al concentrations (i.e. >3–6 mg kg^?1 Al). Although Al-resistant lines could maintain fine root length under these conditions, they were similarly unable to maintain rhizosheath mass. This finding may help to explain why Al-resistant wheats which yield relatively well in deep acid soils, may also benefit from application of lime to the surface layers of the soil.     

环境DNA技术在地下生态学中的应用

地下生态过程是生态系统结构、功能和过程研究中最不确定的因素。由于技术和方法的限制,作为"黑箱"的地下生态系统已经成为限制生态学发展的瓶颈,也是未来生态学发展的主要方向。环境DNA技术,是指从土壤等环境样品中直接提取DNA片段,然后通过DNA测序技术来定性或定量化目标生物,以确定目标生物在生态系统中的分布及功能特征。环境DNA技术已成功用于地下生态过程的研究。目前,环境DNA技术在土壤微生物多样性及其功能方面的研究相对成熟,克服了土壤微生物研究中不能培养的问题,可以有效地分析土壤微生物的群落组成、多样性及空间分布,尤其是宏基因组学技术的发展,使得微生物生态功能方面的研究成为可能;而且,环境DNA技术已经在土壤动物生态学的研究中得到了初步应用,可快速分析土壤动物的多样性及其分布特征,更有效地鉴定出未知的或稀少的物种,鉴定土壤动物类群的幅度较宽;部分研究者通过提取分析土壤中DNA片段信息对生态系统植物多样性及植物分类进行了研究,其结果比传统的植物分类及物种多样性测定更精确,改变了以往对植物群落物种多样性模式的理解。同时,环境DNA技术克服传统根系研究方法中需要洗根、分根、只能测定单物种根系的局限,降低根系研究中细根区分的误差,并探索性地用于细根生物量的研究。主要综述了基于环境DNA技术的分子生物学方法在土壤微生物多样性及功能、土壤动物多样性、地下植物多样性及根系生态等地下生态过程研究中的应用进展。环境DNA技术对于以土壤微生物、土壤动物及地下植物根系为主体的地下生态学过程的研究具有革命性意义,并展现出良好的应用前景。可以预期,分子生物学技术与传统的生态学研究相结合将成为未来地下生态学研究的一个发展趋势。     

The impact of limited soil moisture and waterlogging stress conditions on morphological and anatomical root traits in maize (Zea mays L.) hybrids of different drought tolerance

Effects of soil drought or waterlogging on the morphological traits of the root system and internal root anatomy were studied in maize hybrids of different drought tolerance. The investigations comprised quantitative and qualitative analyses of a developed plant root system through determining the number, length and dry matter of the particular components of the root system and some traits of the anatomical structure of the seminal root. Obtained results have demonstrated a relatively broad variation in the habit of the root system. This mainly refers, to the number, length and dry matter of lateral roots, developed by seminal root, seminal adventitious and nodal roots as well as to some anatomical properties of the stele, cortex and metaxylem elements. Plants grown under waterlogging or drought conditions showed a smaller number and less dry matter of lateral branching than plants grown in control conditions. The harmful effect of waterlogging conditions on the growth of roots was greater when compared with that of plants exposed to drought. In the measurements of the root morphological traits, the effect of soil drought on the internal root anatomical characteristic was weaker than the effect of soil waterlogging. The observed effects of both treatments were more distinct in a drought sensitive hybrid Pioneer D than in drought resistant Pioneer C one. The drought resistant hybrid Pioneer C distinguished by a more extensive rooting and by smaller alterations in the root morphology caused by the stress conditions than drought sensitive hybrid Pioneer D one. Also the differences between the resistant and the sensitive maize hybrids were apparent for examined root anatomical traits. Results confirm that the hybrid Pioneer D of a high drought susceptibility was found to be also more sensitive to periodieal soil water excess. A more efficient water use and a lower shoot to root (S:R) ratio were found to be major reasons for a higher stress resistance of the hybrid Pioneer C. The reasons for a different response of the examined hybrids to the conditions of drought or waterlogging may be a more economical water balance and more favourable relations between the shoot and root dimensions in the drought resistant genotype. The observed modifications of the internal root structure caused by water deficit in plant tissues may partly influence on water conductivity and transport within roots. The results suggest that the morphological and anatomical traits of the maize root system may be used in practice as direct or indirect selection criteria in maize breeding.     

The effects of lime and phosphorus on the function of wheat roots in acid top soils and subsoils

Two wheat varieties with differing aluminium tolerance were grown in pots of acid soil. Liming did not change significantly the amounts of chemically extractable P and K, but caused improved vegetative growth, increased inflow of P and K and reduced uptake of Al. Without lime, roots had a higher content and concentration of P than shoots; liming reversed this. Without lime the sensitive variety with a shorter root length had an Al inflow ten times that of the tolerant one: tolerance involves a mechanism for exlcuding Al. The inflow of P per unit inflow of Al (mol ratio) without lime was three times greater for the tolerant variety which therefore has more P to counteract the effects of Al. The same varieties were grown in two-layer soil columns, with a low P status and a limed topsoil and acid subsoil. Liming the subsoil improved plant growth but this was still restricted by low P availability. Addition of P to the topsoil caused good growth regardless of subsoil acidity: root growth increased in both layers and P (labelled with³²P) taken up from the topsoil was translocated to roots in the subsoil. This P inactivated root Al and allowed the roots to grow and take up more P from the acid subsoil with however a reduction in inflow. The sensitive variety was affected more by the acid subsoil and low P availability, had a similar ability to translocate P to subsoil roots but could not attain the growth rate of the tolerant wheat even with P and lime.     

Tuber aestivum Vittad. mycelium quantified: advantages and limitations of a qPCR approach

A quantitative real-time PCR (qPCR) marker Ta0 with hydrolysis probe (“TaqMan”), targeted to the internal transcribed spacer region of the ribosomal DNA, has been developed for quantification of summer truffle (Tuber aestivum) mycelium. Gene copy concentrations determined by the qPCR were calibrated against pure culture mycelium of T. aestivum, enabling quantification of the mycelium in soil and in host roots from the fields. Significant concentrations of the fungus were observed not only in the finest roots with ectomycorrhizae but also in other root types, indicating that the fungus is an important component of the microbial film at the root surface. The concentration of T. aestivum in soil is relatively high compared to other ectomycorrhizal fungi. To evaluate the reliability of the measurement of the soil mycelium density using qPCR, the steady basal extracellular concentration of the stabilized T. aestivum DNA should be known and taken into account. Therefore, we addressed the stability of the qPCR signal in soil subjected to different treatments. After the field soil was sieved, regardless of whether it was dried/rewetted or not, the T. aestivum DNA was quickly decomposed. It took just about 4 days to reach a steady concentration. This represents a conserved pool of T. aestivum DNA and determines detection limit of the qPCR quantification in our case. When the soil was autoclaved and recolonized by saprotrophic microorganisms, this conserved DNA pool was eliminated and the soil became free of T. aestivum DNA.     

Enumeration of 16S rDNA of Desulfotomaculum lineage 1 in rice field soil by real-time PCR with SybrGreen detection

Real-time PCR is a new and highly sensitive method for the quantification of microbial organisms in environmental samples. This work was conducted to evaluate real-time PCR with SybrGreen (SG) detection as quantification method for Desulfotomaculum lineage 1 organisms in samples of rice field soil. The method was optimized in several parameters like SG concentration. These allowed quantitative PCR with different primer combinations yielding PCR products with lengths up to 1066 bp and with sensitivities of 10(2) targets for all assays. The detection limit in environmental DNA extracts (rice bulk soil and rice roots) was 10(6) targets per gram dry weight according to the dilution of the DNA extracts necessary to overcome PCR inhibition of humic substances. A verification, that the fluorescence increase was due to specific PCR products, was done by agarose gel electrophoresis since melting curve analysis of the PCR products did not show a distinct peak in the first derivative, when the environmental DNA extracts were used in PCR. Amplification with a primer combination specific for Desulfotomaculum lineage 1 organisms showed an abundance of this group of approximately 2% and 0.5% of the eubacterial 16S rDNA targets in rice bulk soil and rice root samples, respectively. Approximately half of this number was obtained in both habitats with a PCR assay specific for a Desulfotomaculum sequence cluster obtained previously from rice field soil.     

Influence of pasture management (nitrogen and lime addition and insecticide treatment) on soil organisms and pasture root system dynamics in the field

At an upland field site in Scotland on an established Festuca-Agrostis pasture, the effects of soil amendment on root dynamics, using nitrogen and lime and the regular application of insecticide, were studied over a period of 1 year. The most common insect root herbivore at the site was Tipula paludosa, and the application of insecticide (chlorpyrifos) reduced numbers of all insect larvae of all species. Root biomass, root appearance, root disappearance and root density were all reduced by insecticide. This reduced rooting could reflect reduced root replacement, due to the reduction in root herbivory in insecticide-treated plots or could be a direct affect of insecticide application on the roots. Root appearance, root disappearance and C and N input to the soil were increased by treatment with nitrogen and lime, while root survival time was reduced. The nitrogen and lime treatment also increased bacterial numbers in the soil and enhanced their potential C utilization. An altered rooting density and longevity was brought about by the two soil treatments, which could have both direct and indirect effects on the soil biota.     

Unravelling below-ground plant distributions: a real-time polymerase chain reaction method for quantifying species proportions in mixed root samples

Knowledge on below-ground plant distributions is almost lacking to date, despite the fact that such information would be very valuable in understanding below-ground competition and species-specific interactions, processes that are expected to shape community structure. Methods available so far for below-ground species determination have drawbacks that we tried to challenge. Some methods make use of differences in the chemical composition between species, but this is highly variable upon environmental factors. DNA-based techniques - far less dependent on chemical composition - such as polymerase chain reaction on internal transcribed spacer (ITS) primers can so far only determine presence-absence of a species in a mixed root sample. Here, we present a quantitative DNA-based technique that allows investigation of relative species abundances in experimental mixed root samples. We used quantitative real-time polymerase chain reaction (PCR) on species-specific markers obtained from intersimple sequence repeat (ISSR) analyses in root samples. This molecular technique is novel in the field of root ecology and its development overcame three challenges: (i) determination of species-specific DNA fragments, (ii) development and optimization of the real time PCR protocol, (iii) designing a data treatment method based on a modified delta-delta-cycle threshold (CT) analysis. The method gained robustness from using relative DNA abundances in species mixtures rather than absolute concentration readings. This requires accurate multispecies reference series as a calibration. Test samples with different known biomass ratios of all species showed proof of concept of this method. The pro's and contra's of this method are discussed in the light of its contribution to advancing ecological research on below-ground plant-plant interactions.     

Changes in root morphology of white lupin (Lupinus albus L.) and its adaptation to soils with heterogeneous alkaline/acid profiles

The ability of Lupinus albus L. to adapt to a heterogeneous soil profile containing acid subsoil below limed topsoil of the same type, and to utilize nutrients by significantly altering its root system structure, was investigated using specially constructed soil profile tubes. Plants grown in homogeneous acid profiles had the fastest growth while those grown in homogeneous limed-soil profiles showed the slowest growth and exhibited some chlorosis after 19 days. Limed topsoil combined with an acid subsoil profile initially retarded plant growth similar to that in a homogeneous limed soil. However, after 68 days significantly greater growth had occurred in the limed/acid soil treatment relative to the homogeneous limed soil, indicating plants had benefited from the acid subsoil stratum. Plants in the homogeneous limed soil profile had lower concentrations of P, Fe and Mn in shoots compared with those in heterogeneous soils. In contrast, the concentration of Ca increased by 74%, due mainly to an increase in the water-soluble Ca fraction. When grown in a heterogeneous limed/acid soil profile, concentrations of P, Ca, K, Mg, Fe, Mn and Zn in shoots were comparable to those grown in a soil with a homogeneous acid profile. Although total root production was lower in the homogeneous limed-soil profile compared to the acid-soil containing profiles, cluster root mass was maintained at a level comparable with that in acid soil. The roots in heterogeneous soil profiles exhibited extensive plasticity, demonstrating a root-type specific, morphological response to the soil conditions. Within the acid subsoil of a heterogeneous profile, there was a large increase in cluster root mass compared with non-cluster roots. The proliferation of cluster roots in acid soil below limed topsoil may enhance the plant's ability to exploit this soil and facilitate the cultivation of L. albus on limed soil. This revised version was published online in August 2006 with corrections to the Cover Date.     

Quantification of Fusarium graminearum in infected wheat by species specific real-time PCR applying a TaqMan Probe

A new real-time PCR based method was developed for the species-specific detection, identification and quantification of Fusarium graminearum in planta. It utilizes a TaqMan hybridisation probe targeting the beta-tubulin gene and a plasmid standard. The assay is highly specific giving no product with DNA of closely related species. It is very sensitive, detecting down to five gene copies per reaction, and is able to produce reliable quantitative data over a range of six orders of magnitude.