The effect of irrigation , soil cultivation system and nitrogen fertilizer on the vitality and content of selected sugars in Vicia faba seed

In this study the influence of sprinkler irrigation, various soil cultivation systems (conventional, reduced tillage, zero tillage system) and the level (0, 30, 60, 90 kg N/ha) of nitrogen (N) fertilization on the vitality and content of selected sugars in faba bean seeds (Vicia faba L.) of the cultivar Nadwis´lan´ski was examined. Sprinkler irrigation of faba bean improved seed energy and germination in all three years of the study (1999-2001) -on average germination energy by 8.8% and total germination by 3.2%-. Germination of faba bean seed under conventional tillage in the drier years was significantly higher than in the zero tillage system. In the wetter year, seed from both simplified systems produced seeds with higher germination than in traditional conventional tillage. Nitrogen (N) fertilizer affected germination energy, but had no effect on faba bean germination. Sprinkler irrigation and N fertilization had no effect on the content of the sugars studied in the faba bean seed. However, the stachyose content of faba bean seeds from conventional tilled plants was significantly higher than in seed of zero tilled plants (0.78 mg/g seed dm), and the galactose content of seed from zero tilled plants was significantly higher than in the other two cultivation systems - 0.34 and 0.28 mg/g seed dm in seeds from conventional and reduced tillage system, respectively.

The RFOs are important carbon reserves and are readily available on germination.They are considered to be stress metabolites during water deficit and cold (Keller and Pharr, 1996;Górecki et al., 1997a;Peterbauer and Richter, 2001;Pinheiro et al., 2005).High number of hydroxyl groups and high molecular mass are important for the osmoprotective functions of carbohydrates including the RFOs.Verbascose contains seventeen hydroxyl groups, and is an osmoprotectant with high molecular mass (828 Da) (Piotrowicz- Cies ´lak et al., 2007).
According to the literature RFOs are a reserve material which accumulate in seed and fruits (Dey, 1985(Dey, , 1990;;Górecki et al., 1997a,b).Further, they are responsible for the transport of nutrient components in the plant.They also increase tissue tolerance to low temperatures (Larsson et al., 1993;Bachmann et al., 1994).Oligosaccharides give more stable intracellular glasses in dry seed than in mono-and disaccharides, and they correlate with acquisition of desiccation tolerance and seed longevity (Hoekstra et al., 2001).Further, the RFOs detoxicate cells by binding free galactose.The decrease in monosaccharide concentration influences a reduction in respiration intensity and impediment of metabolic processes being a source of free radicals.On the other hand, a low level of cell fructose, glucose and galactose eliminates Maillard's reaction whose products contribute to degradation of enzymatic and structural proteins (Dey, 1985(Dey, , 1990)).
The hypothesis of this study was that agronomic treatment such as sprinkler irrigation, soil cultivation systems and N fertilizer can change the levels of sugars in faba bean seed.The aim of the research was to determine the effect of rainfall and sprinkler irrigation of three soil cultivation systems and four levels of N fertilizer on changes in selected sugars in the seed of the faba cultivar Nadwis ´lan ´ski.

Material and methods
The studies were carried out on faba bean (Vicia faba L. minor Harz.) seed harvested from a field experiments grown in 1999-2001 at the Experimental Farm Zl / otniki near Poznan ´ (Poland) in a randomised block design with four replicates.The first factor was water -sprinkler irrigation or no irrigation; the second factor was soil cultivation system (conventional, reduced tillage or zero tillage system); the third factor was different N fertilizer levels (with N doses of 0, 30, 60 and 90 kg N ha -1 ).
The duration of faba bean vegetative growth, without irrigation was: in 1999, 98 d; in 2000, 113 d; and in 2001, 117 d.Total rainfall was 216 mm, 201 mm and 256 mm, in the respective years.With sprinkler irrigation the faba bean vegetative duration was 110 d in 1999, 121 d in 2000 and 117 d in 2001.The sum of natural rainfall was respectively: 220.7 mm, 250.7 mm and 256 mm.On the sprinkler irrigated plots, in 1999-2000, irrigation was applied f ive times at 40 mm a time giving a total of 200 mm year -1 .In 2001, irrigation was applied three times in doses of 40 mm, 40 mm and 30 mm respectively; a total of 110 mm.The irrigation was applied during the period of highest plant sensitivity to water deficit (from the end of May to the end of July or first days of August -during budding, flowering and beginning of pods setting).The irrigation maintained soil moisture, in the 0-30 cm layer at approximately 70% of field capacity.
Laboratory analyses conducted included seedling value (parameters such as percentage of germination, energy of germination, seed purity, the incidence of seed-borne diseases) and the content of oligosaccharides in the faba bean seed.Energy and germination were determined on seed samples taken from plot.The RFO sugars and their concentration were determined using a mean sample from the four replicates.Germination energy and total germination were determined on 100 seeds from each plot.They were placed in a cuvette on filter paper, at 20°C.The energy value was taken at 5 d and total germination at 14 d following ISTA rules.
The α-galactosides were isolated from the seed according to the method of Muzquiz et al. (1992) with some modif ications.Faba bean flour (0.5 g) was homogenized in 5 mL of 50% ethanol for 1 min at room temperature in an Ultraturrax homogenizer.The mixture was centrifuged for 5 min at 3,000 g.The supernatant was decanted and the procedure was repeated twice.The sample extract was purified using C 18 cartridges (500 mg/6 mL) connected to a vacuum system.The effluent was evaporated to dryness, redissolved in deionized water (1 mL) and centrifuged for 8 min at 6,000 g.Analysis of the RFOs was carried out on the supernatant by high performance liquid chromatography using a Beckman HPLC with a refraction index detector.For oligosaccharide separation a Spherisorb-5-NH 2 column (250 × 4.6 mm i.d.) and acetonitrile/water 60-40 (v/v) were used in the mobile phase.Solvents were filtered through a Millipore FH (0.45 µm) membrane and degassed under a vacuum.The injection volume was 20 µL.Quantification of each sugar was accomplished by comparing peak sample areas with a standard solution over the range of 0-4 mg mL -1 and the coefficients of determination above 0.99.
The results were statistically analyzed, using the variation analysis in STATPAK software.Significant differences was determined using the Tukey test at α = 0.05.Variation analysis of germination was performed after conversion to Bliss values.Relationships among seed characteristics were determined using Pearson's correlation coefficient.

Results
Irrigation had no effect on germination energy in 1999.However, in 2000 and 2001 it was significantly increased (Table 1).In all three years sprinkler irrigation improved the total germination of faba bean seed and particularly in 2000.On average, sprinkler irrigation increased germination energy by 8.8% and total germination by 3.2%.
Overall cultivation systems had no significant effect on germination energy and total germination.Soil cultivation system had no effect on germination energy in 1999.However, in 2000, conventional tillage was significantly better than the other two systems.In year 2001, both simplified systems significantly improved germination energy compared with conventional tillage (Table 1).In 1999 and 2000 total germination was significantly higher in seed from conventionally tilled plants compared with zero tillage.It was different in 2001 when seeds from both simplif ied cultivation systems had significantly higher total germinations than seed from conventionally tilled plants.
On average, over the three years, the highest N dose significantly decreased germination energy compared with the three lower doses.However, there was no significant difference in total germination in response to N dose both for mean values and for all analyzed years.
Among the sugars studied, in all years, the highest content in faba bean seed was sucrose which was 45% of the total sugar content (average over all treatments).Among the oligosaccharides, the highest content, 15%, was stachyose.Verbascose was 10% and raffinose 4% of the total content of the sugars studied.On average, among the oligosaccharides analyzed, neither sprinkler irrigation nor N fertilizer had any effect on the content of the sugars analyzed.However, in all years, cultivation system significantly modified the galactose and stachyose content (Fig. 1).In the case of galactose, seed from zero cultivated plants contained 13% more of this sugar than seed from reduced till plants, and 16.3% more than seed from conventionally tilled plants.The reverse was observed for stachyose concentration, where seed from plants which had simple cultivation system, had a decreased stachyose content.
The fructose content of faba bean seeds depended on the interaction of water with N fertilization.With no irrigation increased N doses had no effect on fructose contents (Table 2).With irrigation, and 30 kg N ha -1 , compared with the control, there was a signif icant increase in seed fructose content.However, with 90 kg N ha -1 there was a significant decrease in fructose content -21.3%-in seed from irrigated plots compared with seed from unirrigated plots.
On the average, the highest seed galactose content was in seed from zero tilled plots.There were significantly lower contents in seed from traditionally and reduced till plots.Under irrigation, the lowest seed galactose content was in seed from conventionally tilled plots.There was a signif icantly higher galactose content in seed from reduced till plots, and the statistically highest galactose content was in seed from zero till plots (Table 2).With no irrigation, seeds from zero till plots contained signif icantly more galactose than seed from conventional and reduced till plots.
With conventional tillage seed from unirrigated plots had a significantly higher galactose level, 13.5%, than seed from irrigated plots.With reduced till, there was a trend for a higher galactose content in seed from unirrigated plots.
Pearson's linear correlation analyses between all analysed sugars are presented in Table 3, and one of the strongest connections is shown in Figure 2.  In a column values followed by the same uppercase letter do not differ.Means in a row followed by the same lowercase letter do not differ at p ≤ 0.05.

Discussion
According to Horbowicz and Obendorf (1994), RFOs have a positive effect on the storing ability of seed generic resources.In this process, the proportion of sucrose to oligosaccharides is a significant factor.It is suggested that seed characterized by the proportion of sucrose to RFOs less than 1.0 lose 50% of their vitality after being stored for over 10 years.When the proportion is greater than 1.0, the period is significantly shorter.In this work, the proportion of sucrose to oligosaccharides was 1.6.Thus it can be suggested that these seeds may lose 50% of their vitality in storage in a period of significantly less than 10 years.
In seed of different legume species, a different content of raffinose homologues is observed.Prusinski (2001a, b) argued that in RFOs accumulation, synthesis of verbascose usually dominates, preceded by accumulation of raffinose and stachyose, which conditions the acquisition of tolerance to seed desiccation.Lahuta et al. (2005a) found that the dominant sugar of mature seed of some species of the genus Vicia genus was verbascose, while raffinose and stachyose occurred in smaller amounts.In this study, among the analyzed αgalactosides raffinose, stachyose and verbascose, the highest amount was stachyose -8.94 mg g -1 (mean value for the three years), which makes it 53.2% of the RFOs studied.In this study, the total content of oligosaccharides in faba bean seed was variable over the years.It ranged from 7.98 mg g -1 in the moist year of 2001 to 18.33 mg g -1 in the dryer year of 1999.
The level of sucrose, raff inose and stachyose decreased during starch accumulation.In contrast, during seed ripening, and particularly at the beginning of desiccation there was a dynamic increase of the amount of seed verbascose.This was correlated with the formation of vigour of the ripening seeds (Lahuta et al., 1993).In our study there was no correlation between verbascose content of mature faba bean seed and their vigour.The results of this work indicate that neither sprinkler irrigation nor N fertilizer had a significant effect on the content of the sugars studied, including the oligosaccharides in faba bean seed.However, cultivation system did influence seed stachyose content.There was a significant increase of the content of this sugar with conventional tillage compared with zero tillage.There was a reverse, statistically confirmed, dependence in the case of galactose, where seed from zero tilled plants contained more galactose than those from conventionally tilled plants.Despite the fact that cultivation systems did not differentiate the germination ability of the faba bean seeds, in 1999 and 2000, conventional tillage was significantly more favourable for germination than zero tillage.
In conclusion, the data presented here show that sprinkler irrigation of faba bean over three years of study improved the germination energy and total germination.In dryer years, the total germination of faba bean seed, under conventional tillage, was significantly higher than in seed from zero tilled plants.On the other hand, in a moister year, seed from both simplified cultivation systems produced seed with a higher total germination than in from the conventional system.Nitrogen fertilizer at 0, 30, 60, 90 kg N ha -1 differentiated the germination energy, but had no effect on total germination.Sprinkler irrigation and N fertilizer had no effect on the content of the sugars studied sugars in the faba bean seed.The stachyose content of faba bean seed from plants grown under conventional tillage was signif icantly higher than in seed from direct seeded plants.On the other hand, the glucose content of seed from direct seeded plants was significantly higher than from the other two cultivation systems.

Figure 1 .
Figure 1.Effect of the tillage system on the content of stachyose and galactose in faba bean seed in 1999-2001 and the means of the 3-years.

Figure 2 .
Figure 2. Relationship between the rhamnose and sucrose content of faba bean seed.

Table 1 .
Germination energy and total germination of faba bean seed depending on year of growth, irrigation, soil cultivation system and nitrogen fertilizer (%) a Letters a-c indicate statistical significance at p ≤ 0.05.

Table 2 .
Effect of the irrigation and nitrogen fertilization on fructose content and effect of the irrigation and soil cultivation system on the galactose content of faba bean seed (mg g -1 seed d.m.)