**2.2.1 Experimental details**

The agronomic rotation was tested under a wide range of situations. Three field trial locations were chosen taking into account Italy's wide latitudinal distribution2. Experimental design was thought to produce oilseed from N. tabacum and from traditional oilseed crops (sunflower, soybean, and rapeseed), used as comparison to validate the methodology. Each field was divided into two parts and B.juncea was sown only in one half of the field. To maximize the biofumigant effect, green manuring with B.juncea biomass was carried out when the crop reached flowering. After this, sowing of soybean, sunflower and rapeseed as well as the transplant of tobacco plantlets took place in both parts of the field. In order to make the proposal as flexible as possible, four different fertilization treatments were used: low input (30 kg/ha of chemical fertilizer3), medium input (90 kg/ha of chemical fertilizer), high input (140 kg/ha of chemical fertilizer) or organic input (10000 kg/ha of poultry manure). Untreated plots were set up as control. All field tests were conducted under Good Experimental Practices (GEP).

To evaluate the effect of the green manure of *B.juncea* on nematode infection, countings of *Meloidogyne* spp. were carried out on soil samples taken from both sides of the field while effects on yield of crops grown in succession were monitored recording the fresh weight per hectare (kg/ha) of plant biomass from both sides of the field. Since the green manure of *B.juncea* supplies organic matter to soil, possibly increasing also its sulphur content, it's relevant to ensure that crops grown after this agronomical practice are not enriched in sulphur and therefore less suitable for biodiesel production4.To check this, sulphur quantification in sunflower seeds and oil were done. Seed samples were taken from the unfertilized plots of both sides of the field, and sulphur content detected by ICP-MS (Inductively Coupled Plasma Mass Spectrometry).

#### **2.2.2 Results and discussion on agronomical aspects**

Research on alternative biofuel aims to face the increasing demand for energy requirements by means of a more sustainable energy supply. From this point of view, greenhouse gases saving is expected from biofuels.

The first year of experimentation makes clear that plants grown in succession of *B. juncea*  resulted in higher biomass. This could be due either to the increase in the organic matter content or to the pest control. Indeed, counting of nematodes revealed a strong effect of the green manure of *B.juncea* on nematode control. The average number of larvae found was almost four times lower in the presence of the biofumigant crop. The use of *B. juncea* as green manure does not influence the sulphur content in sunflower seeds and oil, suggesting no sulphur accumulation occurs in succeeding crops.

In order to assess the chemical properties of *B. juncea* oil for biodiesel destination, the authors quantified the total sulphur, nitrogen and phosphorus content in oil from commercial seeds of *B. juncea*. In table 1 data of the quantifications are reported.

<sup>2</sup> Altedo (BO), Vaccolino (FE) and Santa Margherita di Savoia (FG).

<sup>3</sup> Urea (Nitrogen 46%)

<sup>4</sup> The contents of this element in the final product must be under 10 ppm (UNI EN 14214 - Automotive fuels. Fatty acid methyl esters (FAME) for diesel engines. Requirements and test methods).

control and the progressive increase of organic matter should make the soil eligible again for

The agronomic rotation was tested under a wide range of situations. Three field trial locations were chosen taking into account Italy's wide latitudinal distribution2. Experimental design was thought to produce oilseed from N. tabacum and from traditional oilseed crops (sunflower, soybean, and rapeseed), used as comparison to validate the methodology. Each field was divided into two parts and B.juncea was sown only in one half of the field. To maximize the biofumigant effect, green manuring with B.juncea biomass was carried out when the crop reached flowering. After this, sowing of soybean, sunflower and rapeseed as well as the transplant of tobacco plantlets took place in both parts of the field. In order to make the proposal as flexible as possible, four different fertilization treatments were used: low input (30 kg/ha of chemical fertilizer3), medium input (90 kg/ha of chemical fertilizer), high input (140 kg/ha of chemical fertilizer) or organic input (10000 kg/ha of poultry manure). Untreated plots were set up as control. All field tests were conducted

To evaluate the effect of the green manure of *B.juncea* on nematode infection, countings of *Meloidogyne* spp. were carried out on soil samples taken from both sides of the field while effects on yield of crops grown in succession were monitored recording the fresh weight per hectare (kg/ha) of plant biomass from both sides of the field. Since the green manure of *B.juncea* supplies organic matter to soil, possibly increasing also its sulphur content, it's relevant to ensure that crops grown after this agronomical practice are not enriched in sulphur and therefore less suitable for biodiesel production4.To check this, sulphur quantification in sunflower seeds and oil were done. Seed samples were taken from the unfertilized plots of both sides of the field, and sulphur content detected by ICP-MS

Research on alternative biofuel aims to face the increasing demand for energy requirements by means of a more sustainable energy supply. From this point of view, greenhouse gases

The first year of experimentation makes clear that plants grown in succession of *B. juncea*  resulted in higher biomass. This could be due either to the increase in the organic matter content or to the pest control. Indeed, counting of nematodes revealed a strong effect of the green manure of *B.juncea* on nematode control. The average number of larvae found was almost four times lower in the presence of the biofumigant crop. The use of *B. juncea* as green manure does not influence the sulphur content in sunflower seeds and oil, suggesting

In order to assess the chemical properties of *B. juncea* oil for biodiesel destination, the authors quantified the total sulphur, nitrogen and phosphorus content in oil from

4 The contents of this element in the final product must be under 10 ppm (UNI EN 14214 - Automotive

commercial seeds of *B. juncea*. In table 1 data of the quantifications are reported.

fuels. Fatty acid methyl esters (FAME) for diesel engines. Requirements and test methods).

quality productions.

**2.2.1 Experimental details** 

under Good Experimental Practices (GEP).

(Inductively Coupled Plasma Mass Spectrometry).

saving is expected from biofuels.

3

Urea (Nitrogen 46%)

**2.2.2 Results and discussion on agronomical aspects** 

no sulphur accumulation occurs in succeeding crops.

2 Altedo (BO), Vaccolino (FE) and Santa Margherita di Savoia (FG).


Table 1. Nitrogen, sulphur and phosphor content in *B. juncea* oil.

In table 2 the mean percentage increasing of biomass of *B. napus*, *H. annus*, *G. max*, and *N*. *tabacum* produced after green manuring of *B. juncea* is summarized.


Table 2. Increasing of biomass of oilseed crops produced after green manuring of B. *juncea.*
