Tara Singh Gahoonia

Root traits as tools for creating phosphorus efficient crop varieties

This paper provides a brief assessment of the genetic variation in root properties (root morphology, including root hairs), mycorrhizal symbiosis, uptake kinetics parameters and root-induced changes (pH, organic acids and acid phosphatase) in the rhizosphere of various crop species and their genotypes and then briefly discusses the opportunities and challenges of using such knowledge for enhancing P efficiency of future crop genotypes by genetic means. Wide genotypic variation and heritability of root morphology, root hair length and density and thereby P acquisition provide opportunities for selection and breeding for root characteristics for increasing P acquisition. The progress is challenged by the concerns of high carbon cost of larger root systems and by the lack of cost effective methods to determine root length of a large number of genotypes under field conditions. The carbon cost of root hairs is low. Furthermore, low cost methods now exist to compare root hair formation of field grown genotypes. The development and application of sophisticated methods has advanced our knowledge on the role of mycorrhizal symbiosis in P acquisition and also on the molecular basis of fungi and plant interactions. However, extensive studies to explore genotypic variation in mycorrhizal responsiveness are rare, which makes it difficult to assess, how mycorrhizal symbiosis can be manipulated through breeding efforts. The promising variation found in P uptake kinetics parameters of crop genotypes in few studies indicates that more genotypes may be screened by relatively simple nutrient solution culture techniques. The genetic manipulation of the overall differences in cation-anion uptake, which is the main cause of rhizosphere pH change, may be difficult. For manipulation of rhizosphere pH, agronomic measures such as applications of ammonium or nitrate fertilisers may be more useful than breeding approaches. Also it seems difficult to assess what kind of genetic analysis should be performed to support the breeding efforts. Phosphorus mobilisation effect of pH depends on soil P compounds, therefore will differ with soil type. Both the enhanced release of organic acids and higher acid phosphatase activity in the rhizosphere may be useful for increasing P acquisition from inorganic and organic P pools, respectively. Modification of these traits by genetic means should be considered. For successful breeding programmes, the role of various root traits needs to be targeted in an integrated manner and then methods need to be developed for studying their importance under natural soil conditions, so that the genotypic variation can be explored and their ecological significance in P acquisition can be established.

Root hairs and phosphorus acquisition of wheat and barley cultivars

Root-soil contact is an important factor for uptake of a less mobile soil nutrient such as phosphorus (P) by crop plants. Root hairs can substantially increase root-soil contact. Identification of crop cultivars with more and longer root hairs can, therefore, be useful for increasing P uptake in low input agriculture. We studied the root hairs of wheat (Triticum aestivum L. cvs. Kosack, Foreman, Kraka) and barley cultivars (Hordeum vulgare L. cvs. Angora, Hamu, Alexis, Canut) in relation to P depletion from the rhizosphere in three soils of different P levels (0.45, 1.1 and 1.6 mmoles P kg−1 soil; extracted with 0.5 M NaHCO3). Root hairs were measured in solution culture having nutrients and concentration similar to soil solution. Root hairs of Kraka were much longer (1.27 ± 0.26 mm) and denser (38 ± 3) hairs mm−1 root) than those of Kosack which had shorter (0.49 ± 0.2 mm) and fewer (24 ± 3) hairs mm−1 root) root hairs. Root hairs increased root surface area (RSA) of Kraka by 341%. The increase with Foreman was 142% and with Kosack it was 95%. For winter barley, the length (1.1 ± 0.3 mm) and density (30 ± 1 hairs mm−1 root) of root hairs of Hamu differed from root hair length (0.52 ± 0.18 mm) and density (27 ± 1 hairs mm−1 root) of Angora. Root hairs of spring barley cultivars differed in length (Canut 1.0 ± 0.24 mm; Alexis 0.64 ± 0.19 mm) but not in density (Canut 31 ± 1, Alexis 30 ± 2 hairs mm−1 root). Root hair diameter (12 ± 1µm) did not differ among the cultivars. Root hairs increased RSA of Canut by 245%, Hamu by 237%, Alexis by 143% and Angora 112%. The variation in root hair parameters of the cultivars was related to quantity of P depleted from rhizosphere. The correlation (R2) between the root hair lengths of wheat cultivars and the quantity of P depleted from the rhizosphere soil (Q) was (0.99***) in low-P, (0.85***) in medium-P and (0.78**) in high-P soil. The values of (R2) between the root hair surface areas of wheat cultivars and Q were (1.00***) in low-P, (0.74**) in medium-P and (0.66**) in high-P soil. Similar high values of R2 were found for barley. These results show that the variation in root hairs of cereal cultivars can be considerable and it can play a significant role in P acquisition, especially in low-P soils.

Mobilization of phosphate in different soils by ryegrass supplied with ammonium or nitrate

Mobilization of soil P as the result of plant-induced changes of soil pH in the vicinity of plant roots was studied. Seedlings of ryegrass were grown in small containers separating roots from soil by a 30-μm meshed nylon screen which root hairs could penetrate but not roots. Two soils were used, a luvisol containing P mainly bound to calcium and an oxisol containing P mainly bound (adsorbed) to iron and aluminum. Plant-induced changes of soil pH were brought about by application of ammonium-or nitrate-nitrogen. After plants had grown for 10 d the soil was sliced in thin layers parallel to the root mat which had developed on the screen, and both soil pH and residual P determined. Mobilization of P was assessed by P-depletion profiles of the rhizosphere soil.Soil pH at the root surface decreased by up to 1.6 units as the result of ammonium N nutrition and it increased by up to 0.6 units as the result of nitrate N nutrition. These changes extended to a distance between 1 and 4 mm from the root surface depending on the type of soil and the source and level of nitrogen applied. In the luvisol, compared to zero-N treatment, P mobilization increased with the NH4-induced decrease in pH, whereas the NO3-induced pH increase had no effect. In contrast, in the oxisol a similar pH decrease caused by NH4 nutrition had no effect, whereas the pH increase caused by NO3 increased markedly the mobilization of soil P. It is concluded that in the luvisol calcium phosphates were dissolved by acidification, whereas in the oxisol adsorbed phosphate was mobilized by ligand exchange.

The effects of root-induced pH changes on the depletion of inorganic and organic phosphorus in the rhizosphere

A new method allowing control of rhizosphere pH and mineral nutrition was applied to study depletion of various organic and inorganic phosphorus fractions extractable sequentially with 0.5M KHCO3 (pH 8.5), 0.1M NaOH and residual P extractable with 6M H2SO4 from the rhizosphere soil.Soil pH was affected about 2 mm from the root mat. Depletion zones of inorganic P (KHCO3-Pi) extractable with 0.5M KHCO3 extended up to about 4 mm but the depletion zones of all other P fractions were about 1 mm only. The root-induced decrease of soil pH from 6.7 to 5.5 increased the depletion of total P from all fractions by 20% and depletion of KHCO3-Pi and residual P by 34% and 43%, respectively. Depletion of organic P (KHCO3-Po) extractable with 0.5M KHCO3 was not affected by a change in rhizosphere pH. With constant or increased pH, depletion of inorganic P (NaOH-Pi) was 17% and organic P (NaOH-Po) was 22% higher than with decreased pH. Only 54–60% of total P withdrawn from all fractions was from KHCO3-Pi. Substantial amounts of KHCO3-Po and NaOH-Po were mineralized and withdrawn from the rhizosphere within 1 mm from the root mat, as 11–15% of total P withdrawn originated from the organic P fractions. A remaining 11–16% was derived from NaOH-Pi, and 15–18% from residual P fractions likely to be rather immobile. Thus, 40–46% of the P withdrawn near the root mat of rape originated from non-mobile P fractions normally not included in 0.5M NaHCO3 extraction used to obtain an index of plant-available soil P.

Variation in root hairs of barley cultivars doubled soil phosphorus uptake

Length and density (number mm-1 root) of root hairs of two barley (Hordeum vulgare L.) cultivars Salka and Zita and their capability to absorb phosphorus (P) from nutrient solution as well as from rhizosphere soil were studied. The cultivars were chosen because they differed most among 30 cultivars in ability to absorb P from low P soil in two field conditions. In nutrient solution culture, Salka had 32±4 root hairs mm-1 root, 1.02±0.22 mm long. Zita had 21±3 hairs mm-1 root, 0.54±0.14 mm long. In soil, the root hairs of both the cultivars were slightly longer (Salka 1.10 ±0.16 mm; Zita 0.63±0.18 mm) than in solution culture but the difference was non-significant (p<0.05). The root hairs increased the effective root surface area of Salka by 206% and that of Zita by 81%. In solution culture, Salka produced 163±9 m g-1 and Zita 153±11 m g-1 dry roots in 21 days. Salka produced 1.65±0.22 g and Zita 1.51±0.31 g of green dry matter (DM). The cultivars did not differ in uptake of P from nutrient solution culture. The P content of DM was 0.42±0.1% in Salka and 0.41±0.08% in Zita. In soil, Salka depleted two times more P from rhizosphere than Zita. The longer root hairs of Salka increased the extension of the depletion zone for NaHCO3-Pi (inorganic P extracted with 0.5 M NaHCO3) in the rhizosphere. The cultivars also depleted NaOH-Pi (inorganic P extracted with 0.1 M NaOH) from the rhizosphere soil, but the difference between the cultivars was non-significant (p<0.05). The results suggested that the ability of Salka to absorb more inorganic soil P was due to its longer and denser root hairs.

Direct evidence on participation of root hairs in phosphorus (32P) uptake from soil

Root hairs substantially extend root surface for ion uptake. Although many reports suggest a relationship between root hairs and phosphorus (P) uptake of plants, the role of root hairs in phosphorus uptake from soils is still debated. We measured uptake of phosphorus from soil directly via root hairs. Root hairs only were allowed to penetrate through a tightly stretched nylon screen (53 µm) glued to the bottom of a PVC tube. The penetrating root hairs grew for 2 and 4 days in soil labelled with radioisotope phosphorus (P) tracer 32P (185 kBq g-1 dry soil) filled in another PVC tube. Transparent plastic rings of thickness ranging from 0.25 mm to 2.0 mm were inserted between the two PVC tubes. This provided slit width for microscopic observations in situ, which confirmed that only root hairs were growing into the 32P labelled soil. In some cases no rings were inserted (slit width = 0) where both root hairs and root surface were in contact with the labelled soil (total 32P uptake). The uptake of32 P from soil via the root hairs only was quantified by measuring activity of 32P in the plant shoot (32P uptake only via root hairs).The results showed that when 70 percent of the root hairs grew into the labelled soil, they contributed to 63 percent of the total P uptake. With decreasing number of root hairs growing into the 32P labelled soil, the quantity of 32P in the plant shoot decreased. In this study, P uptake via root hairs was measured in a soil-based system, where root hairs were the only pathway of 32P from soil to the plant shoot. Therefore, this study provides a strong evidence on the substantial participation of root hairs in uptake of phosphorus from soil.

A root hairless barley mutant for elucidating genetic of root hairs and phosphorus uptake

This paper reports a new barley mutant missing root hairs. The mutant was spontaneously discovered among the population of wild type (Pallas, a spring barley cultivar), producing normal, 0.8 mm long root hairs. We have called the mutant bald root barley (brb). Root anatomical studies confirmed the lack of root hairs on mutant roots. Amplified Fragment Length Polymorphism (AFLP) analyses of the genomes of the mutant and Pallas supported that the brb mutant has its genetic background in Pallas. The segregation ratio of selfed F2 plants, resulting from mutant and Pallas outcross, was 1:3 (−root hairs:+root hairs), suggesting a monogenic recessive mode of inheritance.In rhizosphere studies, Pallas absorbed nearly two times more phosphorus (P) than the mutant. Most of available inorganic P in the root hair zone (0.8 mm) of Pallas was depleted, as indicated by the uniform P depletion profile near its roots. The acid phosphatase (Apase) activity near the roots of Pallas was higher and Pallas mobilised more organic P in the rhizosphere than the mutant. The higher Apase activity near Pallas roots also suggests a link between root hair formation and rhizosphere Apase activity. Hence, root hairs are important for increasing plant P uptake of inorganic as well as mobilisation of organic P in soils.Laboratory, pot and field studies showed that barley cultivars with longer root hairs (1.10 mm), extracted more P from rhizosphere soil, absorbed more P in low-P field (Olsen P=14 mg P kg−1 soil), and produced more shoot biomass than shorter root hair cultivars (0.63 mm). Especially in low-P soil, the differences in root hair length and P uptake among the cultivars were significantly larger. Based on the results, the perspectives of genetic analysis of root hairs and their importance in P uptake and field performance of cereals are discussed.

Microbial community composition and functional diversity in the rhizosphere of maize

This study investigates the small-scale stratification of bacterial community composition and functional diversity in the rhizosphere of maize. Maize seedlings were grown in a microcosm with a horizontal mesh (53 μM) creating a planar root mat and rhizosphere soil. An unplanted microcosm served as control. Thin slices of soil were cut at different distances from the mesh surface (0.2–5.0 mm) and analysed for bacterial community composition by PCR-DGGE (polymerase chain reaction-denaturing gradient gel electrophoresis) of 16S rDNA and tested for activities of different enzymes involved in C, N, P and S cycling. Bacterial community composition and microbial functional diversity were affected by the presence of the maize roots. The bacterial composition showed a clear gradient up to 2.2 mm from the root surface, while no such gradient was observed in the unplanted pot. Invertase and phosphatase activities were higher in the close vicinity of maize roots (0.2–0.8 mm), whereas xylanase activity was unaffected. This study shows that the changes in bacterial community composition and functional diversity induced by roots may extend several millimetres into the soil.

Phosphorus (P) acquisition of cereal cultivars in the field at three levels of P fertilization

Low phosphorus (P) availability in soils and diminishing P reserves emphasize the need to create plants that are more efficient P users. Knowledge of P efficient germplasm among the existing cereal varieties may serve as the basis for improving soil P use by selection and breeding. We had identified some cereal cultivars (winter wheat: Kosack and Kraka; winter barley: Hamu and Angora; spring barley: Canut, Alexis, Salka, Zita;) which differed (p<0.05) in P depletion from thin slices (0.2 mm) of the rhizosphere soil under controlled conditions. In the present study, the same cultivars were studied under field conditions at three levels of P supply (no-P, 10 and 20 kg P ha-1) and the differences in P uptake as found in the previous work were confirmed. Under both conditions, the variation between the cultivars was greatest in soil without P fertilizers (no-P) for about 30 years. The variation in P uptake with most cultivars disappeared when 10 kg P ha-1 was applied. Root development did not differ between the cultivars much, but there was wide, consistent variation in their root hairs, regardless of growth media (solution, soil column and field). Increase in soil P level reduced the length of root hairs. The variation in root hairs between the cultivars was largest in no-P soil. When 10 kg P ha-1 was applied, the root hair lengths did not differ between the cultivars. Barley cultivars with longer root hairs depleted more P from the rhizosphere soil and also absorbed more P in the field. The relationship between root hairs and phosphorus uptake of the wheat cultivars was less clear. The wide variation in P uptake among the barley cultivars in the field and its relationship to the root hair development confirms that root hair length may be a suitable plant characteristic to use as criterion for selecting barley cultivars for P efficiency, especially in low-P soils.

Root-released organic acids and phosphorus uptake of two barley cultivars in laboratory and field experiments

A major portion of phosphorus (P) applied as fertilizers is bound in soils as P compounds of variable adsorption strength, reducing the effectiveness of P fertilization. Plant genotypes equipped with mechanisms for utilizing the adsorbed P more efficiently can, therefore, enhance the effectiveness of P fertilization. Such genotypes will also enrich plant gene pools for further analysis and upgrading of P efficiency by selection and breeding. We studied the variation and the mechanisms of P uptake of two winter barley (Hordeum6ulgare L.) cultivars Marinka and Sonate (parents of existing 200 haploid progeny lines), by laboratory and field experiments. After cultivation in nutrient solution for 21 days, Marinka produced more roots than Sonate, but similar amounts of dry shoots of lower P content (Marinka 3.49 0.4 mg g 1 , Sonate 4.990.6 mg g 1 ). The total P uptake per plant did not differ between the cultivars. Marinka retained more P in roots as indicated by the higher concentration of P in the roots (Marinka 3.99 0.3 mg g 1 and Sonate 3.090.4 mg g 1 ). In sterile nutrient solution culture, the cultivars differed mainly in release of organic acids from the roots, with Marinka releasing three times more citric acid and nearly two times more acetic acid than Sonate. The cultivars had similar root hair lengths and they did not differ (P\ 0.05) in depletion of available soil P fraction (extracted with 0.5 M NaHCO3) in the rhizosphere. Marinka absorbed nearly twice as much P from the strongly adsorbed soil P fraction (extracted with 0.1M NaOH). Also under field conditions, Marinka absorbed more P and produced more shoot dry matter. The higher P uptake by Marinka than Sonate can be attributed to its ability to acquire P from strongly adsorbed soil P by releasing more organic acids, especially citric acid, from its roots.