Use of 33P to trace in situ the fate of canola below-ground phosphorus, including wheat uptake in two contrasting soils

Our understanding of the contribution of crop root residues to phosphorus (P) cycling is mainly derived from studies using excavated roots re-introduced to soil. This study aims to quantify total below-ground P (BGP) of mature canola in situ and to estimate directly the proportion accessed by subsequent wheat. 33P-Labelled phosphoric acid was fed by stem wick to canola (Brassica napus) grown in sand or loam in pots. Shoots were removed from all plants at maturity. Half of the pots were destructively sampled. After a 3-week fallow, wheat was grown for 5 weeks in the remaining undisturbed pots. At canola maturity, 23–36% of the 33P was partitioned in recovered roots and 34–40% in the soil. More 33P was recovered in the loam than the sand. Within the soil, 6–10%of the fed 33P was present in resin P and 3–5%was in hexanol-released P pools. Ratios of shoot P : BGP(8 : 1 in sand and 15 : 1 in loam) were much narrower than those of shoot P : recovered root P (17 : 1 in sand and 39 : 1 in loam). A greater proportion and amount of the mature canola BG33P was recovered by wheat grown in the loam (26%, 2.6 mg/plant) than in the sand (21%, 1.5 mg/plant). The majority of canolaBG33P remained in the bulk soil. Input of P below-ground by mature canola and subsequent P benefit to wheat was greater in loam than sand. The P from canola below-ground residues contributed up to 20% of P uptake in wheat during the first 5 weeks of growth. Longer term benefits of P from below-ground residues require investigation.Foyjunnessa, C, Ann McNeill, Ashlea Doolette, Sean Mason, and Mike J. McLaughli

Isotope studies of accumulation and cycling of phosphorus and nitrogen below-ground in canola and lupin

It is commonly acknowledged that the cycling of nutrients, including phosphorus (P) and nitrogen (N), from plant residues in crop rotations is important for the sustainability of agricultural systems. This is especially the case for Australian low input rain-fed cropping systems, where, due to economic, climatic and edaphic factors, additions of P and N as fertilizers or manures are limited. Optimal management of P and N cycled from break crop residues requires a sound understanding of the quantity of each nutrient in residues and what proportion potentially becomes available for a following cereal crop. A review of the literature (Thesis Chapter 1) highlighted that whilst there is information concerning quantities of N, and to a lesser extent P, contained in mature above-ground crop residues, much less has been reported concerning quantities of P or N of below-ground (BG) residues from various crop species. This is partly because root studies are time consuming and hence expensive to undertake, but also quantification is hampered by the certainty that not all roots can be recovered from soils, especially in fine textured soils. As a result root turnover and nutrient release have largely been investigated under somewhat ‘artificial’ or ‘unrealistic’ conditions - using roots that have been extracted from soil, dried, often chopped or finely ground and finally incorporated back into soil to decompose. More recent innovative studies, summarised in the review (Chapter 1), have used a stem wick-feeding technique to label crop root systems in situ with the 15N isotope. These studies demonstrated that total BG N accumulation for these crop species was larger than quantified from recovered roots alone. The labelling technique allowed for direct in situ quantitative tracing of the N from legume and oilseed root residues into subsequent wheat plants. It was demonstrated that up to 20% of wheat N uptake may be derived from the BG N input by root systems of a previous break crop. The review (Chapter 1) further highlighted that quantitative assessment of the amounts of P accumulated by crop root systems were extremely scarce and there did not appear to be any in situ isotope studies related to P accumulation BG. Hence the work described in this thesis broadly explored the potential to adapt the approaches used for ¹⁵N isotope studies in order to quantitatively assess in situ P accumulation BG by break crop species in soils differing in texture, and the uptake of P derived from those BG break crop residues by a following wheat plant. The specific aims of the work were: i) to adapt the stem wick-feeding technique for use with ³³P to allow in situ quantification of total BG P accumulation by plants, ii) to quantify and compare BG P in two break crops species (an oilseed and a legume) important in Australian rain-fed cropping systems, iii) to assess and measure whether soil texture influences BG P accumulation in canola (oilseed) and lupin (legume), and iv) to trace the fate of break crop BG P relative to BG N in a following cereal (wheat). Preliminary assessment of methodologies used in estimation of BG N in crop plants and their suitability for ³³P studies for BG P were undertaken (Thesis Chapter 2). It was found that the ‘dry’ method frequently used to recover roots for isotope studies (viz: freeze dry manually picked roots with adhering soil, brush roots clean) was comparable to the conventional ‘wet’ root recovery method (viz: washing soil from roots over a sieve), in that similar amounts of root were recovered, which did not differ in P concentration and were not contaminated by soil. Recovery and measurement of roots from field soil cores suggested the amount of P in canola roots in the topsoil (to 0.1m) could be as much as 4 kg ha⁻¹ compared to 1.5 kg ha⁻¹ for rye and less than 1 kg ha⁻¹ for lupin. Other preliminary studies identified that in stem wick-fed plants, ³³P isotope activity was lower where soil P availability (manipulated by P fertiliser addition) was greater. However, the feeding technique could be used to effectively label root systems of lupin with ³³P even at a late vegetative stage of plant growth when it might be considered that the shoot would be the primary sink for P redistributed within the plant. A further study (Chapter 3; Paper 1) confirmed that a substantial proportion (26-51%) of wick-fed ³³P was allocated to recoverable roots of canola and lupin grown in sand. Since this first main study did not detect any ³³P in soil, a mass balance approach was used to determine the amount of unrecovered ³³P, which was suggested to be largely present in unrecovered fine roots, designated as root-derived (RD) P. Using this indirect approach it was estimated that RD P represented 15% of total BG P for canola and 32% for lupin. A subsequent study in deeper pots (Chapter 4; Paper 2) fed a larger amount of ³³P and extended scintillation counting time for samples to improve the method detection limit. This facilitated the direct estimation of unrecovered RD P for canola and lupin at late vegetative stage in two contrasting soil textures, sand and loam. Estimated total BG P accumulation by both crop species was at least twice that of recovered root P and was a greater proportion of total plant P for lupin than canola. There was more unrecovered RD P in the loam than the sand within each species. No ³³P was detected in labile P pools (resin-P or hexanol released-microbial P) at this late vegetative stage of sampling which suggested that there had been no active efflux of ³³P-labelled orthophosphate from labelled roots or any root turnover. However, from a subsequent study (Chapter 5; Paper 3) where ³³P labelled canola plants were sampled at maturity it was evident that after the late vegetative stage root turnover may occur, with 3-5% of fed ³³P detected in the hexanol-released pool and 6-10% in the resin P pool– the higher values being for a loam textured soil which contained a higher proportioned of the fed ³³P than the sand. There appeared to be no translocation of P from roots to shoot between late vegetative stage and maturity since the proportion of fed ³³P recovered BG was the same (70%) at both times. The proportion and amount of canola BG ³³P that was recovered in subsequently grown wheat was higher in the loam (26%; 2.6 mg P) than sand (22%; 1.5 mg P) reflecting the larger pool of BG P in the loam and the faster turnover rate of BG residues. However, this P derived from the previous crop BG residues represented an equal proportion (20%) of the total wheat P uptake in both soils (Chapter 5, Paper 3) since wheat dry matter production was less in the sand. Hence the P benefit from the previous plant BG residues was the same for wheat on both soils. Dual feeding with ³³P and ¹⁵N was used in the final study reported in this thesis (Chapter 6; Paper 4) to simultaneously assess in situ (i) BG N and BG P accumulation by mature lupin and canola, and (ii) the relative contribution from the decomposition of these BG residues to the N and P nutrition of following wheat. The hypothesis tested was that P release from canola BG residues would be relatively greater than from lupin BG residues whereas N release would be relatively smaller. Partitioning of fed ¹⁵N differed from ³³P with the majority of fed ¹⁵N recovered in shoots while a larger proportion of fed ³³P was allocated BG. The amount of total BG P was greater for canola than lupin although lupin had a higher amount of total BG N (75 mg N plant⁻¹) than canola 68 mg N plant⁻¹). C:P ratio of lupin roots was 708:1 and 188:1 for canola. Root C:N ratio was 39:1 for canola and 24:1 for lupin. The N:P ratio for lupin roots was wider (29:1) than canola (5:1), but the N:P ratio of the RD fractions was similar (6:1 canola; 7:1 lupin). Proportion of BG P taken up by wheat was significantly, but only slightly greater after canola (21%) than after lupin (19%), and since BG P was greater for canola this represented 20% of total wheat P uptake and 12% for wheat after lupin. Despite larger lupin BG N, a lower proportion (~8%) was taken up by wheat than from canola BG N (~12%) and so contribution to wheat total N uptake by lupin BG residues (~10%) was surprisingly less than from canola (12.5%). It was concluded from this final study that P uptake by wheat from residues was related to total BG P of the residues but not total BG N. The proportion of P and N from BG residues of mature canola and lupin taken up by wheat did not appear driven by C:P or C:N ratio of recovered roots, but by P concentration of roots, and possibly N:P ratio of BG residues. Research presented in this thesis demonstrates significantly greater amounts of P in BG residues compared to those previously estimated using root recovery methods alone, and that about one-third of total plant P may be partitioned BG. Thus potential P and N benefits to wheat from cycling of break crop root residues are likely to be more substantial than currently thought, and potentially comparable to contributions from an annual P fertilizer addition in low input rain-fed systems. Results further suggest an interaction between release of N and P from BG residues, with an apparent P limitation to the release of N by lupin BG residues; hence C to nutrient ratio of roots was not a good predictor of nutrient release. Lastly, this research also highlights the contribution by root residues of break crops to the longer term fertility of soils, since a large proportion of the BG P and N remains in soil after wheat. In summary, this work develops greater quantitative understanding of the direct contribution of the BG P and BG N of canola and lupin to wheat in terms of P and N supply, and a greater understanding of P and N accumulation in break crop roots. The adaptation of the stem wick-feeding technique for in situ ³³P-labelling of plants opens up exciting future research opportunities in determining the accumulation, fate and interactions of break crop BG P and BG N under undisturbed conditions in following cereals.Thesis (Ph.D.) (Research by Publication) -- University of Adelaide, School of Agriculture, Food and Wine, 2016

Dual-labelling (15N and 33P) provides insights into stoichiometry and release of nitrogen and phosphorus from in situ mature lupin and canola below-ground residues

Background and aims: Belowground (BG) residues may contribute significant amounts of N and P via nutrient cycling to following crops, particularly in low fertiliser input systems or where all the above-ground residues are removed. Reports of simultaneous measures of nitrogen (N) and phosphorus (P) release from mature crop residues, especially from intact root systems in situ, are rare. A single stem-feed of ¹⁵N followed by ³³P was used to (i) estimate total amounts of N and P accumulated in situ BG by mature canola and lupin plants including the stoichiometry of N and P in recovered and non-recovered components of these BG residues, and ii) simultaneously trace and quantify the relative release of that N and P. Methods: One set of pots were destructively sampled at lupin and canola maturity to quantify total accumulation of BG P and BG N, including estimated N and P in unrecovered roots plus root-derived materials (RD N and RD P). Shoots were removed from a second set of pots into which wheat was sown after a 3 weeks fallow. Release of P and N from the decomposing in situ ³³P/¹⁵N labelled lupin and canola BG residues was assessed as uptake in 5 weeks old wheat. Results: Canola root had higher P and lower N concentrations than lupin. Canola total BG P was greater than lupin with a higher proportion as estimated RD P. Estimated RD N was similar in both species but lupin had more N in roots and so higher total BG N. C:P ratio of lupin roots was 708:1 and 188:1 for canola. Root C:N ratio was 39:1 for canola and 24:1 for lupin. N:P ratio for lupin roots was wider (29:1) than canola (5:1), but N:P ratio of RD fractions was similar (6:1 canola; 7:1 lupin). Proportion of BG P released and taken up by wheat was 21% after canola and 19% after lupin, and since total amount of BG P was much greater for canola the quantity of P released was double that after lupin. Proportion of lupin BG N (37%) released was similar to that for canola BG N (33%) although a larger amount of N was released from lupin given the larger BG N pool. Conclusion: The proportion of P and N released from in situ BG residues of mature canola and lupin and taken up by wheat in this short term study was broadly inversely related to C:P, C:N and N:P ratio of recovered roots but results suggest a likely influence also of N:P ratio of the unrecovered BG residues. Quantities of N and P released were a function of the estimated total amount of plant N and P accumulated in situ BG.Foyjunnessa, Ann McNeill, Ashlea Doolette, Sean Maso

Quantifying total phosphorus accumulation below-ground by canola and lupin plants using ³³P-labelling

Background and aims: Measures of phosphorus (P) in roots recovered from soil underestimate total P accumulation below-ground by crop species since they do not account for P in unrecovered (e.g., fine) root materials. ³³P-labelling of plant root systems may allow more accurate estimation of below-ground P input by plants. Methods: Using a stem wick-feeding technique ³³P-labelled phosphoric acid was fed in situ to canola (Brassica napus) and lupin (Lupinus angustifolius) grown in sand or loam soils in sealed pots. Results: Recovery of ³³P was 93% in the plant-soil system and 7% was sorbed to the wick. Significantly more ³³P was allocated below-ground than to shoots for both species with 59-90% of ³³P measured in recovered roots plus bulk and rhizosphere soil. ³³P in recovered roots was higher in canola than lupin regardless of soil type. The proportion of ³³P detected in soil was greater for lupin than canola grown in sand and loam (37 and 73% lupin, 20 and 23% canola, respectively). Estimated total below-ground P accumulation by both species was at least twice that of recovered root P and was a greater proportion of total plant P for lupin than canola. Conclusion: Labelling roots using ³³P via stem feeding can empower quantitative estimates of total below-ground plant P and root dry matter accumulation which can improve our understanding of P distribution in soil-plant systems.Foyjunnessa, Ann McNeill, Ashlea Doolette, Sean Mason, Mike J. McLaughli

In situ (33)P-labelling of canola and lupin to estimate total phosphorus accumulation in the root system

Aims Accurate quantification of P accumulated in plant root systems is difficult since fine roots are often not recovered from soil. ³³P-labelling of plant root systems in soil may facilitate estimation of total below-ground P accumulation. Methods Canola (Brassica napus) and lupin (Lupinus angustifolius), grown in sand in sealed pots, were fed 33P-labelled phosphoric acid via the stemusing a wickfeeding technique. Results More ³³P was partitioned to canola roots (51 %) than lupin roots (26 %), although specific activity of roots for the two species was similar (30– 31 kBq ³³Pmg ³¹P⁻¹) since canola roots had higher ³¹P content than lupin roots (4.1 cf 2.3 mg P plant⁻¹). Mean recovery of fed ³³P (250 kBq plant⁻¹) in the whole plant including recovered roots was 84 % 10 days after feeding, and 6 %was sorbed to the wick. Assuming the unrecovered 10 % of ³³P (below the detection limit for soil digestion method used in this study) was within fine roots, then estimated P in unrecovered fine roots represented 15 % of total root system P for canola and 32 % for lupin. Conclusion Wick-feeding ³³P via the stem can effectively label P in roots in situ and facilitate quantitative estimation of total P accumulation by plant root systemsFoyjunnessa, Ann McNeill, Ashlea Doolette, Sean Mason & Mike J. McLaughli

Using a Tri-Isotope (13C, 15N, 33P) Labelling Method to Quantify Rhizodeposition

Belowground (BG) plant resource allocation, including roots and rhizodeposition, is a major source of soil organic matter. Knowledge on the amounts and turnover of BG carbon (C), nitrogen (N), and phosphorus (P) in soil is critical to the understanding of how these elements cycle in soil-plant system. However, the assumptions underlying the quantification and tracking of rhizodeposition using isotope labeling methods have hardly been tested. The main objectives of this chapter were to (i) review the different plant labeling techniques for each of the three elements; (ii) describe a novel method for the simultaneous investigation of C, N, and P rhizodeposition in sand; and (iii) test the methodological assumptions underlying quantification of rhizodeposition. Stable 13C and 15N isotopes were widely used to study rhizodeposition of plants either separately or in combination, while P radioisotopes (32P, 33P) were used to investigate root distribution. The combination of the 13CO2 single-pulse labeling with the simultaneous 15N and 33P cotton-wick stem feeding effectively labeled Canavalia brasiliensis roots and facilitated the estimation of rhizodeposited C, N, and P input from root systems. However, the isotope distribution was uneven within the root system for all three elements. Additionally, we observed a progressive translocation from shoot to roots for 15N and 33P over 15 days after labeling, while the 13C tracer was diluted with newly assimilated non-enriched C compounds over time. Younger root sections also showed higher specific activities (33P/31P) than the older ones. The relatively high 33P radioactivity recovered in sand right away at the first sampling was attributed to an artifact generated by the stem feeding labeling method. Overall, our results suggest that the assumptions underlying the use of isotope methods for studying rhizodeposition are violated, which will affect the extent of quantification of rhizodeposition. The consequences of nonhomogeneous labeling of root segments of different age require further investigation. The use of a time-integrated isotopic composition of the root is recommended to not only account for temporal variation of isotopes but also to improve the method of quantifying plant rhizodeposition