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.
Root proliferation and phosphorus acquisition in response to stratification of soil phosphorus by two contrasting Trifolium subterraneum cultivars
Aims Phosphorus (P) is usually stratified in the topsoil layer under pasture, due to the broadcast application of fertiliser, excreta and leaf-litter deposition on the soil surface, and minimal soil disturbance. The objective of this study was to investigate root proliferation and P acquisition in response to P stratification by comparing two Trifolium subterraneum cultivars with contrasting root morphologies.
Methods Clover micro-swards were grown with deficient, constrained and sufficient P supplied in a topsoil layer overlying a P-deficient subsoil that mimicked the stratification of P that occurs under pasture. Phosphorus labelled with 33P- and 32P-radioisotope tracer was mixed throughout the topsoil and subsoil layers, respectively.
Results The shoot yield and total plant P uptake of the cultivars increased in response to increased topsoil P supply. The length of roots produced by the cultivars was equivalent in each of the P treatments, although the specific root length achieved by the cultivars was substantially different. In the P-constrained and P-sufficient treatments, ~91% and ~ 99% of total plant P was acquired by topsoil roots, respectively. In contrast, subsoil roots acquired 60-74% of total plant P in the P-deficient treatment.
Conclusions Topsoil roots were most important for P acquisition when P was highly stratified, whereas subsoil roots contributed to P acquisition when P was uniformly distributed throughout the P-deficient soil profile. Selection for prolific nutrient-foraging roots, in conjunction with plasticity for subsoil exploration, may improve the P-acquisition efficiency of T. subterraneum genotypes and confer adaptability across a range of soil-P environments.
Variation in root morphology and P acquisition efficiency among Trifolium subterraneum genotypes
Trifolium subterraneum L. is widely grown in the phosphorus (P) deficient soils of southern Australia. However, this pasture legume has a high critical external P requirement and requires frequent applications of P fertiliser to achieve high productivity. Twenty-six genotypes of T. subterraneum were grown to determine: (i) differences in shoot growth and P acquisition under low-P supply; (ii) the root morphological traits important for P acquisition; and (iii) the feasibility of selection among genotypes for these root morphological traits. Micro-swards of each genotype were grown with a topsoil layer that was either moderately P-deficient or had P supplied in excess of the critical requirement for maximum yield; the subsoil layer was P-deficient. Yield and P content of shoots and roots were determined after 5 weeks’ growth, and root samples were assessed for diameter, length and root hair length. All genotypes were equally highly productive when excess P was supplied. However, relative shoot yield in the moderately P-deficient soil ranged from 38-71%. Total root length ranged from 63-129 m pot-1, and was correlated with total plant P uptake (R2 = 0.78, P < 0.001). Variation was also observed in average root diameter (0.29-0.36 mm) and root hair length (0.19-0.33 mm). These traits were combined with root length to calculate the total surface area of the root hair cylinder, which was also correlated with total plant P uptake (R2 = 0.69, P < 0.001). The results demonstrated that there was significant variation in P acquisition efficiency and shoot yield among genotypes of T. subterraneum when grown in P-deficient soil, and that root length was important for improved P uptake. The results indicate potential to identify superior genotypes that achieve improved P acquisition and higher shoot yields in low-P soil.
Root proliferation in response to P stress and space: implications for the study of root acclimation to low P supply and P acquisition efficiency
Aims The experiment was conducted to understand how root morphological traits contributed to the contrasting P-acquisition efficiencies of two Trifolium subterraneum cultivars.
Methods Phosphorus acquisition, root length proliferation and root acclimation to P deficiency were investigated by varying the P supply (20-300 mg P kg−1) and planting density (1-12 plants pot−1) of clover micro-swards.
Results The shoot yield of both cultivars increased in response to increased P supply and increased planting density. Higher planting densities increased shoot yield by reducing the time to leaf canopy closure. In P-deficient soil, the ‘less P-efficient’ cultivar acquired less P and yielded relatively poorly compared with the ‘more P-efficient’ cultivar when planting density was low (<6 plants pot−1). However, the P acquisition and yields of the cultivars were equivalent when planting density was high (~6-12 plants pot−1). Both cultivars proliferated nutrient-foraging roots in response to P limitation and space (i.e. lower planting densities). However, the proliferation responses of the cultivars differed, particularly in response to space.
Conclusions Differential proliferation responses, combined with differences in specific root length, explained how the cultivars differed in P-acquisition efficiency. The results indicate that inappropriate cultural conditions (e.g. planting densities) may confound attempts to define P-efficient root traits and to identify genotypes with improved P-acquisition efficiency.
Intrinsic root morphology determines the phosphorus acquisition efficiency of five annual pasture legumes irrespective of mycorrhizal colonisation
Mycorrhizal fungi are ubiquitous in agroecosystems and form symbiotic associations that contribute to the phosphorus (P) acquisition of many plants. The impact of mycorrhizas is most pronounced in P-deficient soil and commonly involves modifications to the root morphology of colonised plants. However, the consequences of mycorrhizal colonisation on root acclimation responses to P stress are not well described. Five annual pasture legumes, with differing root morphologies, were grown to determine the effect of mycorrhizal colonisation on shoot yield, root morphology and P uptake. Micro-swards of each legume were established in pots filled with a topsoil layer that had been amended with five rates of P fertiliser. The topsoil overlaid a low-P subsoil that mimicked the stratification of P that occurs under pasture. Mycorrhizal colonisation improved P acquisition and shoot yield in the low-P soil treatments, but did not reduce the critical external P requirement of the legumes for near-maximum yield. The yield responses of the mycorrhizal plants were associated with reduced dry matter allocation to topsoil roots, which meant that the P acquisition benefit associated with mycorrhizal colonisation was not additive in the P-deficient soil. The contribution of the mycorrhizal association to P acquisition was consistent among the legumes when they were compared at an equivalent level of plant P stress, and was most pronounced below a P stress index of ~0.5. The intrinsic root morphology of the legumes determined their differences in P-acquisition efficiency irrespective of mycorrhizal colonisation.
Foraging for better root traits: phosphorus acquisition efficiency in a critical pasture species
Pastures grown on P-deficient soils in temperate southern Australia use mixtures of grasses and legumes. The main legumes (Trifolium and Medicago spp.) are highly productive across a wide range of environments but have high 'critical' P requirements (i.e. the P supply needed for near-maximum yield) relative to the grasses with which they grow. Improving the P-efficiency of the most important legume (T. subterraneum), or developing the agronomic merit of alternatives that are already P-efficient (e.g. Ornithopus spp.) would deliver reductions in P fertiliser inputs, improve farm incomes, and achieve better use of scarce nutrient resources. Here we describe research to improve the P efficiency of T. subterraneum. Field and controlled-environment experiments, with various pasture legume species, have demonstrated that substantial differences in the nutrient foraging potential of their roots determines their requirement for P fertiliser. Three key root morphology traits ensure efficient P acquisition from low P soil: development of high root length, high specific root length and long root hairs. Ornithopus spp. deploy an "optimal" combination of these root traits, efficiently maximising soil exploration to capture more P and to yield well in low P soils. In contrast, Trifolium subterraneum develops long roots in response to low P but has low specific root length and short root hairs which limit its ability to explore soil for P. Within T. subterraneum, variation exists in specific root length and root proliferation. These key factors determine intra-specific variation in P acquisition with the best genotypes achieving twice the yield of the worst in low P soil. The short root hairs on T. subterraneum (0.2-0.4 mm) are a major factor limiting P acquisition efficiency. Wider studies of nutrient foraging root traits among genetically-allied Trifolium species from the Section Trichocephalum revealed substantial differences in propensity for root foraging (11-35 cm root/cm3 soil) and root hair length (0.2-0.5 mm) but, like T. subterraneum, no genotypes tested to date have root foraging traits in the optimal combinations achieved by Ornithopus spp. To drive further substantive change in the P efficiency in the key pasture legume, T. subterraneum, it will be necessary to break through apparent intra-specific 'boundaries' for specific root length and root hair length by identifying radical ecotypic outliers, inter-specific introgression or directed mutagenesis.
Differences in subsoil P acquisition by two subterranean clover cultivars in a P deficient soil
Phosphorus (P) is usually concentrated in the uppermost layers of the soil profile under pasture, hence topsoil root allocation is important for maximising P acquisition. However, total root length was recently found to be a marginally better predictor of variation in P uptake among twenty-six genotypes of subterranean clover (Trifolium subterraneum L.) when compared to topsoil root length alone. This result prompted a preliminary assessment of P acquisition by subsoil roots. Micro-swards of two cultivars were grown with a topsoil layer that was either P-deficient or amended with P for improved plant growth, overlying a low-P subsoil that contained 32P-labelled phosphate. Both cultivars produced less shoot dry mass under P constraint, and the cultivar that allocated more root length to the subsoil layer produced a larger shoot dry mass in the P-deficient soil. This cultivar also recovered more 32P-labelled phosphate from the subsoil layer in both P treatments. Therefore, variation exists for subsoil P acquisition and this trait may be important for determining shoot yield in P-deficient soil.