The utilization of nitrogen by sugarcane (Saccharum spp.) fertilized with sunn hemp (SH)(Crotalaria juncea L.) and ammonium sulfate (AS) was evaluated using the 15N tracer technique in the experiment 2, that consisted of four treatments with four replications in a randomized block design as fallow: a) control with no N fertilizer or green manure; b) ammonium sulfate (AS) at a rate of 70 kg ha-1 N; c) sunn hemp (SH) green manure; d) and sunn hemp plus ammonium sulfate (SH + AS). Microplots consisting of three rows of sugarcane 2-m long were set up in plots c and d with the 15N-labeled sunn hemp.

N was added at the rate of 196 and 70 kg ha-1 as 15N labeled sunn hemp green manure (SH) and as ammonium sulfate (AS), respectively. Treatments were: (i) Control; (ii) AS15N; (iii) SH15N + AS; (iv) SH15N; and (v) AS15N + SH. Sugarcane was cultivated for five years and was harvested three times. 15N recovery was evaluated in the two first harvests.

Sunn hemp (Crotalaria juncea L, cv IAC-1) was sown at the rate of 25 seeds per meter on the 4 Dec 2000 and emerged in nine days. Microplots, consisting of 6 rows, 2- m long and spaced by 0.5 m within the sunn hemp plots were used for 15N enrichment as described by Ambrosano [24]. After 79 days the sunn hemp was cut, and the fresh material was laid down on the soil surface. Total dry mass of sunn hemp was equivalent to 9.15 Mg ha-1, containing 21.4 g kg-1 N, corresponding to 195.8 kg ha-1 N with an 15N enrichment of 2.412 atoms % excess.

Microplots with AS-labeled fertilizer (3.01 ± 0.01 atoms % 15N), with two contiguous rows 1­m long, were set up in plots b and also in plots d; therefore, these plots had microplots for both sunn hemp and AS-labeled materials.

Ammonium sulfate was sidedressed to sugarcane 90 days after planting in both main plots and microplots. N rate (70 kg ha-1) is within the range (30 to 90 kg ha-1 N) recommended for the plant cane cycle in Brazil [25]. A basal fertilization containing 100 kg ha-1 P2O5 as triple superphosphate and 100 kg ha-1 K2O as potassium chloride was applied to all treatments to ensure a full sugarcane development. Cane yield was determined outside the microplots by weighing the stalks of three rows of sugarcane, 2-m long.

Stalks yields were measured after 18 months (plant-cane cycle, on 24 Aug 2002), 31 months (1st ratoon crop, on 8 Oct 2003), and 43 months after planting (2nd ratoon crop, on 20 Sep 2004). Samples consisting of ten stalks were used for the determination of apparent sucrose content (Pol) in the cane juice, according to [23]. The expressed cane juice was analyzed for Pol (apparent sucrose) by a saccharimeter. Just before harvesting of the plant cane (24 Aug 2002) and of the first ratoon (8 Oct 2003) whole plants were collected from 1-m row of plants in the center of the microplots. Leaves and stalks were analyzed separately for determination of 15N abundance and N content in a mass spectrometer coupled to an N analyzer, following the methods described in [26].

The fraction and amount of nitrogen in the plant derived from the labeled source (Ndff) and the fraction of N recovery of the labeled source (R%) were calculated based on the isotopic results (atoms %), according to Trivelin [26], Equations 1 to 3:

where: Ndff (%) is the fraction of nitrogen in the plant derived from the labeled source, a and b are 15N abundance values (atoms % excess) in the plant and in the labeled source (AS or SH), respectively; QNdff (kg ha-1) is the amount of nitrogen in the plant derived from the labeled source, TN (kg ha-1) is total cumulative nitrogen in the sugarcane plant (kg ha-1 ); R% is the fraction of N recovery of the labeled Sugarcane cultivar IAC — 87-3396 was planted on Mar or April on plots with ten sugarcane rows, 10-m long and spaced at 1.4 m.

The biological nitrogen fixation (BNF) by leguminous plants was determined by natural abundance of 15N technique (S15N) [27], and sunflower was the non-N fixing specie. The chemical analysis of plants to determine macro and micronutrient contents were performed according to the methods proposed by [28].