**2. Materials and methods**

In this review, the results from three separate field trials, set in different years and locations, are reported and discussed. Accordingly, the three experiments are described separately.

#### **2.1 HEAR adaptation to reducing agricultural inputs**

The trial was carried out for 2 years during growing seasons 2006-07 and 2007-08 (autumn sowing) in Legnaro (45°21'N, 11°58'E, NE Italy) at the experimental farm of the University

Adaptability and Sustainable Management

detector (FID) (275°C).

**Soil tillage** 

*Brassica carinata*.

**Spring N fertilisation (kg ha-1)** 

Sweden) on 2 g of milled seeds, with diethyl ether as solvent.

of High-Erucic *Brassicaceae* in Mediterranean Environment 105

During stem elongation and flowering stages, at about 7-day intervals, the N status of the crop was checked by determining the shoot nitrogen content (Kjeldahl method). Harvesting took place about in mid-June in 2007 (Hearty and Marcant June 5, Maplus June 11), and later in 2008 ( June 19, all genotypes), by a combine harvester equipped with a wheat-cutting bar. Seed oil content was measured by the Soxtec-Tecator equipment (FOSS Analytical, Höganäs,

The profiles of fatty acids were determined on methylated oil (preparation of methyl esters according to International Standard ISO 5509) (Bondioli & Della Bella, 2002) by gaschromatography (GC 1000, DANI Instruments, Milan – Italy). Helium was used as carrier gas and a capillary column (SUPELCO SPTM-2380, 30 m long, 0.25 mm outer diameter, 0.20 µm film) to separate fatty acids. The oven was set at an initial temperature of 180°C and maintained for 5 min; later, 240°C was reached by progressive increases (+5°C per minute) and maintained for 8 min, so that each analysis lasted 25 min. The gas chromatograph had a split/splitless (SL/IN) injector (250°C) with a split rate of 100:1 and a flame ionization

**2.2** *Brassica carinata* **response to N fertilization in multi-locality trials set in NE-Italy**  In order to test the possible introduction of *B. carinata* in NE Italy, extensive field trials were set during 2006-2009 in two different locations, both in the Veneto region (NE Italy). The two sites were Legnaro (45°21'N, 11°58'E), at the University of Padova experimental farm, and Rosolina (45°04'N, 12°14'E), at the experimental research center of the Veneto region called "Pò di Tramontana". The sites were only 35 km apart, but they were characterised by different soil type, climatic conditions, and agronomic management (Tab. 4). The N fertilisation strategy was differently applied in the two sites; in the soil at Rosolina,

**Localities LEGNARO ROSOLINA Years** 2006-2008 2007-2009 **Soil type** Silt-loam Sandy-loam **Mean annual temperature** 10.2°C 14.2°C **Mean annual precipitation** 820 mm 639 mm

**Pre-sowing fertilisation (kg ha-1)** 30-90-90 N-P2O5-K2O 30-90-90 N-P2O5-K2O **Sowing date** End of September End of September **Inter-row distance** 0.45 m 0.45 m **Weed management** Mechanical Chemical **Sowing density** 4.5 kg 4.5 kg

**Harvesting date** Mid-June Mid-June Table 4. Most important protocol details of experiments in Legnaro and Rosolina with

Subsoiling or cultivator + disk harrowing + rotary harrowing

> 3 doses: *0 N 100N Reglette dose*

Spading + rotary harrowing

2 doses:

*Low*: 70÷100 N *High*: 100÷140 N

of Padova, following a split-plot experimental design with 3 replicates. Aiming at mimicking farm-scale cultivation as closely as possible, plot size was set at around 400 m2 surface area and agricultural practices were applied by means of farm-scale technologies. Three commercial genotypes of *B. napus* HEAR – two open pollinated varieties: Maplus (NPZ Lembke, Germany) and Hearty (Monsanto, France), and one CHH (Composite Hybrid Hybrid) Marcant (NPZ Lembke, Germany) – were sown on 27 and 28 September respectively in 2006 and 2007 in a silty-loam soil (pH 8.4; 2.5% organic matter; 0.1% total N) by a cereal seeder. The seed dose was 4.5 kg ha-1, roughly corresponding to a density of 100 seeds per m2 for all varieties. Two contrasting levels of inputs, high and low, were tested, involving soil tillage, fertilisation and weed management (Table 2).


Table 2. Characteristics of two contrasting agricultural managements for HEAR rapeseed.

For proper mechanical weed control, the inter-row distance was increased from 0.3 to 0.45 m in the low input regime. After pre-sowing fertilisation (30 kg ha-1 N, for high input only), spring nitrogen supply was calculated following the method proposed by CETIOM (Centre Technique Interprofessionnel des Oléagineux Métropolitains, 1998), called "Réglette Azote". In brief, it recommends increasing doses of N for rising potential yields (i.e., revealed by higher shoot biomass at the end of winter); for the same value of expected yield, the lower the shoot biomass, the higher the amount of N. According to this approach, about at the end of January, the shoot biomass of 1 m2 for each plot (2 replicates per plot) was sampled and weighed (fresh green matter). Biomass values were related to potential yields of 3 and 4 t ha-1, chosen respectively for low and high input managements, to calculate the N dose. The resulting amount of N, applied as ammonium sulphate in mid-February, ranged between 0-20 kg ha-1 in the first year and 0-60 kg ha-1 in the second for low input, and between 35-50 kg ha-1 (first year) and 80-130 kg ha-1 (second year) for high input, depending on genotype (Table 3).


Table 3. Amount of spring nitrogen in two years of trial, calculated by following "Réglette Azote" method: experimental site was considered to have deep soil rich in organic matter.

of Padova, following a split-plot experimental design with 3 replicates. Aiming at mimicking farm-scale cultivation as closely as possible, plot size was set at around 400 m2 surface area and agricultural practices were applied by means of farm-scale technologies. Three commercial genotypes of *B. napus* HEAR – two open pollinated varieties: Maplus (NPZ Lembke, Germany) and Hearty (Monsanto, France), and one CHH (Composite Hybrid Hybrid) Marcant (NPZ Lembke, Germany) – were sown on 27 and 28 September respectively in 2006 and 2007 in a silty-loam soil (pH 8.4; 2.5% organic matter; 0.1% total N) by a cereal seeder. The seed dose was 4.5 kg ha-1, roughly corresponding to a density of 100 seeds per m2 for all varieties. Two contrasting levels of inputs, high and low, were tested,

(pre-emergence)

N = 30 P2O5 = 90 K2O = 90

Table 2. Characteristics of two contrasting agricultural managements for HEAR rapeseed.

and 80-130 kg ha-1 (second year) for high input, depending on genotype (Table 3).

Maplus High 50 130

Hearty High 50 110

Marcant High 35 80

Table 3. Amount of spring nitrogen in two years of trial, calculated by following "Réglette Azote" method: experimental site was considered to have deep soil rich in organic matter.

**N (kg ha-1)** 

Low 0 60

Low 20 40

Low 0 0

**Genotype Input level 2006-07** 

For proper mechanical weed control, the inter-row distance was increased from 0.3 to 0.45 m in the low input regime. After pre-sowing fertilisation (30 kg ha-1 N, for high input only), spring nitrogen supply was calculated following the method proposed by CETIOM (Centre Technique Interprofessionnel des Oléagineux Métropolitains, 1998), called "Réglette Azote". In brief, it recommends increasing doses of N for rising potential yields (i.e., revealed by higher shoot biomass at the end of winter); for the same value of expected yield, the lower the shoot biomass, the higher the amount of N. According to this approach, about at the end of January, the shoot biomass of 1 m2 for each plot (2 replicates per plot) was sampled and weighed (fresh green matter). Biomass values were related to potential yields of 3 and 4 t ha-1, chosen respectively for low and high input managements, to calculate the N dose. The resulting amount of N, applied as ammonium sulphate in mid-February, ranged between 0-20 kg ha-1 in the first year and 0-60 kg ha-1 in the second for low input, and between 35-50 kg ha-1 (first year)

**Inter-row distance** 0.30 m 0.45 m

**High input** L**ow input** 

grubbing + harrowing Disc-harrowing + rotary tillage

Mechanical weeding (one autumn passage)

> N = 0 P2O5 = 50 K2O = 50

> > **2007-08 N (kg ha-1)**

involving soil tillage, fertilisation and weed management (Table 2).

**Soil tillage** Ploughing to 0.35 m depth +

**Weed management** Metazachlor 1,250 g ha-1

**Pre-sowing fertilisation (kg ha-1)** 

During stem elongation and flowering stages, at about 7-day intervals, the N status of the crop was checked by determining the shoot nitrogen content (Kjeldahl method). Harvesting took place about in mid-June in 2007 (Hearty and Marcant June 5, Maplus June 11), and later in 2008 ( June 19, all genotypes), by a combine harvester equipped with a wheat-cutting bar. Seed oil content was measured by the Soxtec-Tecator equipment (FOSS Analytical, Höganäs, Sweden) on 2 g of milled seeds, with diethyl ether as solvent.

The profiles of fatty acids were determined on methylated oil (preparation of methyl esters according to International Standard ISO 5509) (Bondioli & Della Bella, 2002) by gaschromatography (GC 1000, DANI Instruments, Milan – Italy). Helium was used as carrier gas and a capillary column (SUPELCO SPTM-2380, 30 m long, 0.25 mm outer diameter, 0.20 µm film) to separate fatty acids. The oven was set at an initial temperature of 180°C and maintained for 5 min; later, 240°C was reached by progressive increases (+5°C per minute) and maintained for 8 min, so that each analysis lasted 25 min. The gas chromatograph had a split/splitless (SL/IN) injector (250°C) with a split rate of 100:1 and a flame ionization detector (FID) (275°C).
