4.1. Field Site
The experiment was undertaken at the P demonstration plots at the International Rice Research Institute (IRRI), Los Baños, The Philippines, from January–April 2013. The six 0.0135 ha plots (three +P, three −P) contained soil in bunkers transported from a P-deficient site at Pangil, Laguna, The Philippines. Briefly, soil in the −P plots had a pH (1:1 soil-water) 7.7, % C 2.34, % N 0.175, Bray P 1.6 mg·kg−1, exchangeable K (meq·100 g−1) 0.44, and available Zn 1.9 mg·kg−1, while the +P soil had a pH (1:1 soil-water) 7.3, % C 2.33, % N 0.192, Bray P 8.5 mg·kg−1, exchangeable K (meq·100 g−1) 0.54, and available Zn 0.9 mg·kg−1. On 4th January 2013, three days prior to transplanting, basal fertiliser was broadcast on the plots at rates (kg·ha−1): 90 N, 26 P (+P plots only), 33 K, and 2 Zn.
4.2. Plant Cultivation
Rice (cv. IR64) seeds were sown into trays containing commercial nursery seedling mix in a glasshouse at IRRI. At 21 days after sowing (DAS), the 7th January 2013 seedlings were transplanted by hand into the field plots. Seedlings were transplanted in rows 0.2 m apart and a hill spacing of 0.2 m within rows (i.e., 25 hills·m−2), with one plant per hill. A further 90 kg·ha−1 N fertiliser was applied (broadcast) in three splits of 30 kg·ha−1 N at 20, 40, and 60 days after transplanting. Plants were cultivated under standard, fully flooded practice, with weeds controlled by hand weeding.
Anthesis occurred on the 18th March and 28th March for +P plots and −P plots, respectively, and plants reached physiological maturity 28 days after anthesis (DAA), regardless of P treatment. Three plants per plot were harvested at anthesis and at physiological maturity, by severing plant shoots 10 mm above the soil surface. Plant material was dried in an oven at 60 °C for 5 days, and then separated into grain (husk plus caryopsis), stem, flag leaf, second and third leaves, older leaves, and late tillers (tillers that had not yet reached anthesis). Any grains from late-emerging tillers that may not have been physiologically mature at the time of harvest, but were heavy enough to be separated from chaff during in the threshing operation, were recorded in the grain yield.
Samples were analysed for nutrient concentration at Environmental Analysis Laboratories, Lismore, NSW, Australia. A 0.2 g subsample of finely ground tissue was digested with nitric acid in a MARS microwave oven (CEM Corp., Matthews, NC, USA), and concentrations of P, K, Mg, Ca, Fe, Zn, Mn, and Cu in the digest solutions were quantified using inductively coupled plasma optical emission spectroscopy (ICP-OES 4300D, Perkin Elmer, Waltham, MA, USA). Tissue N and S concentrations were measured using a LECO TruMAC CNS analyser.
Grain lysophospholipids were measured as per Liu et al. [15
]. In brief, the ground rice grain (15 mg) was extracted with 75% n-propanol (0.8 mL), and the extract was analysed using liquid chromatography mass spectrometry (LCMS) to quantify the ten major lysophospholipids in rice grain. The concentrations of these lysophospholipids were summed to obtain the total lysophospholipid concentration. Phytate was measured using the method of Shi et al. [16
]. Briefly, ground rice grain was extracted using 0.4 M HCl, and the phytate in the extract was precipitated with an acidic iron-III-solution of known iron content. The phytate concentration was quantified by measuring the decrease of iron in the supernatant using colourimetry.
4.4. Statistical Analyses
Total nutrient content, % post anthesis accumulation, harvest index, grain lysophospholipid and phytic acid concentrations, and tissue concentration data for each nutrient were analysed using a one-way analysis of variance fitting P treatment (plus or minus P fertiliser) in Genstat. Because of the inherent high variability in field studies, a probability level of 0.1 was used: significance of differences between treatment mean values for each trait was tested using Duncan’s multiple range test (p ≤ 0.1).