EFFECT OF SULPHUR ON THE YIELD AND CONTENT OF GLUCOSINOLATES IN SPRING OILSEED RAPE SEEDS

Piotr Mirosław Szulc, Lucyna Drozdowska, Piotr Kachlicki

ABSTRACT

The aim of the present research was to define the effect of sulphur nutrition on the seed yield of ‘Star’ spring oilseed rape and the content of glucosinolates. Sulphur was applied in different forms (elemental or ionic), in different ways (through soil or leaves) and doses (0, 20, 60 kgˇha^-1). The laboratory research material involved seeds from a three-year field experiment set up on degraded black earth containing 1.93 mg SO₄ˇkg^-1 of soil. The content of glucosinolates in seeds was determined with the HPLC according EN ISO 9167-1. The application method and sulphur forms and doses increased the oilseed rape seed yield. The highest mean seed yield was obtained from plants exposed to foliar fertilisation with elemental sulphur at the dose of 60 kgˇha^-1. In this case the content of glucosinolates amounted to 10.78 µMˇg^-1 of dry matter and was lower as compared to objects non-fertilised with sulphur. A total content of glucosinolat

Key words: oilseed rape, fertilisation, sulphur, glucosinolates.

INTRODUCTION

The yield of spring oilseed rape seeds and chemical composition depend on the cultivar, environmental conditions, nitrogen fertilisation and sowing date [11,14]. The oilseed rape cultivation technology also depends much on sulphur fertilisation, especially in the regions of deficit of this macroelement [23].

Wielebski and Wójtowicz [22] observed for winter oilseed rape that the seed yield of 3.5 tˇha^-1 requires 88 kg of sulphur. Schnug and Haneklaus [15] showed that the sulphur uptake by oilseed rape took place especially in spring from the beginning of vegetation period to the end of flowering at the amount of 15-20 kgˇt^-1 of seeds produced. According to these authors, fertilising plants with this macroelement enhances the yield, content of fat and sulphur aminoacids. However it can also increase a total content of glucosinolates in seeds [2,8,16,24]. Glucosinolates are secondary metabolites containing sulphur, generally occurring in Brassicaceae family plants. They are non-toxic substances; however the products of their enzymatic break-down are biologically active compounds. The animal feeding experiments showed severe anti-nutritional effects of the glucosinolate derived compounds, so their level decides about the seeds technological value and the feed value of m eal [7,13]. Both the sowing seeds and seeds for consumption of double-improved oilseed rape cultivars must comply with the Polish standard: 15 µM glucosinolates per g of seeds for sowing seeds and 25 µM per g of defatted seed meal for seeds to be processed [19].

The present research aimed at:

• evaluation of the effect of sulphur on the total glucosinolates content as well as their qualitative and quantitative composition in oilseed rape seeds,

• assessment of the influence of different methods, forms and doses of sulphur application on the seed yield with the optimal share of glucosinolates.

Bearing in mind the essential role of sulphur in oilseed rape agro-ecosystem and metabolism, a working hypothesis has been formulated which assumes that similarly as for winter oilseed rape, sulphur fertilisation will affect yielding and utility value of spring oilseed rape.

MATERIAL AND METHODS

The results which cover the seed yield and glucosinolates content were obtained from a three-year (1997-1999) field experiment of the Department of Plant Cultivation of the Faculty of Agriculture, the Bydgoszcz University of Technology and Agriculture. The experiment was set up as a split-plot-split-block in four replications on degraded black earth of a wheat defective complex. A complete content of sulphur in soil ranged from 0.03 to 0.08% S_total and the mean content of sulphate sulphur amounted to 1.93 mg SO₄ˇkg^-1 of soil. The other soil properties are given in Table 1.

‘Star’ spring oilseed rape was sown at the beginning of the second decade of April on experimental plots of 18 m². In each research year sugar beet constituted a forecrop for spring oilseed rape. NPK
(N = 120 kgˇha^-1, P₂O₅ = 60 kgˇha^-1, K₂O = 120 kgˇha^-1) fertilisation was applied pre-sowing.

Elemental sulphur was obtained from a fungicide preparation, known under brand name of ‘Siarkol’, while crystalline Na₂SO₄ was used as the source of ionic sulphur. Foliar fertilisation involved the usage of 6.7% water solution of Siarkol or Na₂SO₄. The dose of 20 kg Sˇha^-1 was applied before oilseed rape sowing (soil fertilisation) or over the rosette phase (foliar fertilisation). As for plant fertilisation with a dose of 60 kg Sˇha^-1, it was divided into three parts (Table 2). The control was made up of plots not fertilised with sulphur.

The date of two-phase seed harvest coincided with a turn of the first and second decade of August. The seed yield was measured and the seed content of alkenyl glucosinolates (progoitrin, gluconapin, glucobrassicanapin, napoleiferin) and indolyl glucosinolates (4-hydroxyglucobrassicin, glucobrassicin) were analysed using the HPLC of desulphoglucosinolates according to the EN ISO 9167-1 method. The chromatographic analysis was carried out using Merck Hitachi HPLC instrument with Spherisorb ODS2 £ 5 µm (250 × 4.6 mm) column.

The seed yield results and the content of glucosinolates in seeds were verified with variance analysis in a fixed model. The synthesis of the variance analysis was carried out in a mixed model. Mean values for the combination both for the seed yield and the content of glucosinolates were compared with a multiple Tukey test at p = 95%. In a search for variation in the values of the characteristics researched in objects fertilised with sulphur against the control (without sulphur), the Dunnet test was applied.

RESULTS

The observed effects of sulphur application methods, forms and doses on the spring oilseed rape yield varied over the successive research years (Table 3). The highest seed yield, 4.06 and 4.01 tˇha^-1, was obtained in 1997 following the foliar fertilisation with sulphur at the dose of 60 kgˇha^-1 in ionic or elemental form, respectively, and it was 0.76-0.71 tˇha^-1 higher than the seed yield from the control plots. Over 1998-1999 oilseed rape seed yields from all the plots covered by the fertilisation combinations were lower as compared with those of 1997, however, the highest yields were recorded for oilseed rape foliar-fertilised with sulphur in elemental form at the dose of 60 kgˇha^-1. Mean seed yield obtained over 1997-1999 for that experiment variant amounted to 3.23 tˇha^-1 and was 0.81 tˇha^-1 higher than the control. Variance analysis for mean seed yields confirmed the effect of the application metho d on oilseed rape yield and showed that foliar fertilisation was more effective. Comparing the yields obtained from the control and those fertilised sulphur, it was noticed that an increase in the sulphur dose from 20 to 60 kgˇha^-1 increased the yield significantly. Foliar fertilisation of plants with ionic sulphur was the only one for which increasing the dose from 20 to 60 kgˇha^-1 decreased the yield. An interaction was also shown between the application method and fertilisation form. As for soil fertilisation, the plants grown on plots fertilised with ionic sulphur yielded significantly higher than those with elemental sulphur. Foliar fertilisation with sulphur, however, showed better results when sulphur was applied in elemental form.

Sulphur fertilisation modified also the content of glucosinolates in spring oilseed rape which differed over years (Table 4). The total content of glucosinolates in seeds was highest in the first year of the experiment and significantly depended on the fertilisation form and dose. However only foliar fertilisation with ionic sulphur at the dose of 60 kgˇha^-1 increased the content of these compounds as compared with the control. Over the second experiment year all the factors studied as well as their interaction significantly affected the content of glucosinolates in seeds. Sulphur applied into soil increased the total content of glucosinolates as compared with the control; the highest content of glucosinolates was observed in seeds of plants fertilised with sulphur introduced into soil in elemental form. The foliar application of sulphur, irrespective of the form, did not increase the content of these compounds and it remained at the level of the control or was lower. The fertilisation with sulphur in the ionic form resulted in a lower content of glucosinolates then that observed in seeds of plants fertilised with elemental sulphur. In case of soil fertilisation with the elemental form of sulphur an increase of its dose did not influence the content of glucosinolates. In the other fertilisation variants a significantly higher total glucosinolate contents were recorded in seeds collected from plants fertilised with 20 kg Sˇha^-1 as compared to the sulphur dose of 60 kgˇha^-1. The 1998 most favourable fertilisation variant coincided with foliar fertilisation with elemental and ionic sulphur at the dose of 60 kgˇha^-1 which did not increase the content of glucosinolates in seeds. The analysis of seeds collected in the 1999 experiment showed that the content of glucosinolates in seeds, just like in 1998, was significantly differentiated by the factors studied. The highest content of glucosinolates was observed in seeds of plants foliar- fertilised with ionic sulphur. An increase in sulphur dose from 20 to 60 kgˇha^-1 irrespective of the application method significantly increased the total content of glucosinolates when the ionic form was applied. In that research year the most favourable fertilisation method was foliar fertilisation with elemental sulphur and soil fertilisation with ionic sulphur, which did not increase significantly the content of glucosinolates as compared with the control. Variance analysis which covered the three-year research period showed a significant effect of the interaction between the application method and the sulphur form applied on the total content of glucosinolates. The highest content of glucosinolates was recorded in seeds collected from objects soil-fertilised with elemental sulphur. However, there was noted no increased content of the compounds researched in seeds due to soil fertilisation with ionic sulphur and to foliar fertilisation with elemental sulphur.

The factors studied affected the total content of alkenyl glucosinolates (Table 5); over successive experiment years the relationships were similar as for the total content of glucosinolates. Three-year mean results showed an increased content of alkenyl glucosinolates as compared with the control due to soil fertilisation with elemental sulphur and foliar fertilisation with ionic sulphur. However, an increase in the sum of alkenyl glucosinolates was observed neither in seeds of plants soil-fertilised with ionic sulphur nor with foliar-applied elemental sulphur. Out of all the researched alkenyl glucosinolates in seeds, progoitrin dominated; its content, as compared with the control, was significantly higher in seeds obtained from all the experiment variants and was significantly affected by the interaction between two factors: the application method and fertilisation form. Its highest content was noted in seeds collected from plants fertilised with elemental sulphur i nto soil. Sulphur fertilisation did not show a significant effect on the content of gluconapin, accounting for the second largest share of alkenyl glucosinolates in seeds. There was shown a significant effect of the interaction between the application method and fertilisation form on the content of glucobrassicanapin and napoleiferin in seeds. As compared with the control, in seeds obtained from all the experiment objects there was recorded a lower content of glucobrassicanapin. Yet a significant increase was recorded in the content of napoleiferin in seeds when soil was fertilised with elemental sulphur and for foliar fertilisation with ionic sulphur. However a significantly lower content of the glucosinolates researched, as compared with the control, was observed in seeds collected from plants foliar-fertilised with elemental sulphur and soil-fertilised with ionic sulphur.

Most of the factors studied also significantly affected the content of indolyl glucosinolates in seeds (Table 6). Over successive research years there were recorded similar relationships as for the sum of glucosinolates and the sum of alkenyl glucosinolates, especially for the application method and fertilisation form. On average over the three-year research period the content of indolyl glucosinolates was highest following soil fertilisation in elemental form. Studying the effect of sulphur fertilisation on the content of respective forms of indolyl glucosinolates, as compared to those of the control seeds, a higher content of 4-hydroksyglucobrassicin was recorded only in seeds of plants fertilised with elemental sulphur at the dose of 20 kgˇha^-1 into soil and foliar-applied ionic sulphur at the dose of 60 kgˇha^-1. However the content of glucobrassicin in seeds obtained from all the fertilisation variants was significantly higher as compared with the control. Foliar fertilisation with elemental sulphur at the dose of 20 kgˇha^-1 was the only one which showed the content of this glucosinolate similar to the control.

Alkenyl glucosinolates accounted for 57.1% of all the glucosinolates in seeds of plants not fertilised with sulphur, while indolyl glucosinolates – 42.9%. Depending on sulphur application variant, the share of alkenyl glucosinolates in the total pool of these metabolites ranged from 56.2 to 61.8%, while that of indolyl glucosinolates from 38.2 to 43.7% (Fig. 1).

DISCUSSION

Spring oilseed rape, similarly to the winter one, shows a high demand for sulphur from the beginning of shooting to silique setting by plants [18]. Its demand ranges from 15-20 kgˇt^-1 of the seeds produced [20]. Many authors [10,12,15,16,22,24] researching the effect of this macroelement on the growth and yielding of oilseed rape, show a necessity of fertilising plants with sulphur.

Sulphur fertilisation applied in the experiment increased the mean seed yield from 6.61 to 33.4% as compared with the control. The highest mean yield in the present experiment was obtained due to foliar fertilisation of oilseed rape with elemental sulphur at the dose of 60 kgˇha^-1. The sulphur form applied not only increased yield but also improved the phytosanitary status, inhibiting the development of fungal pathogens of Alternaria genus, especially Alternaria brassicae and Alternaria brassicicola species [17].

A positive effect of sulphur fertilisation on oilseed rape cultivated on soils deficient in this macroelement was shown as early as in 1968 by Appelqvist [1] who observed that increasing sulphur dose, whenever its deficit is recorded in the medium, to the amount which would satisfy plant nutrient requirements, increased the yield, as compared with the control, by 80-129%. The report by Bilsborrow et al. [2] showed a varied effect of sulphur fertilisation on oilseed rape yield depending on the sulphur content in soil and in the air. The highest yield was recorded for oilseed rape fertilised with 40 kg Sˇha^-1 cultivated on plots of a low sulphur content, and the yield obtained was 65% higher than that from control objects (poor in sulphur). A weak reaction of oilseed rape to fertilisation with sulphur which coincided with a good sulphur supply was observed by Evans et al. [6], Haneklaus et al. [9] and for oilseed rape cultivated in Poland by Wielebski and Muśnicki [21]. Scar ce research into the effect of sulphur on spring oilseed rape showed that as for soil of low sulphur content (14.3 mg S-SO₄ˇkg^-1 of soil) fertilisation with a dose of 12 kg Sˇha^-1 increased the seed yield from 8.14 to 14.7% as compared with the yield from non-fertilised plants [12]. The results, as well as the present results, state that spring oilseed rape shows a lower increase in seed yield due to sulphur fertilisation applied than traditional and double-low winter oilseed rape cultivars.

Fertilisation of oilseed rape with sulphur, besides an increase in seed yield, can deteriorate the seed quality by increasing the content of glucosinolates [2,8,24]. The present experiment shown differences in the level of these metabolites depending on the application method and sulphur form. There was observed a significant increase in the sum of glucosinolates in plant seeds fertilised with elemental sulphur into soil and a lower content of the sum of glucosinolates and the sum of alkenyl and indolyl glucosinolates in seeds of plants foliar-fertilised with this sulphur form, yet no significant effect of the sulphur dose. The reaction of oilseed rape cultivars to foliar fertilisation with elemental sulphur was researched by Booth et al. [4], showing that fertilising the ‘double-low’ cultivars with a dose of 16 kg Sˇha^-1 increased only the sum of indolyl glucosinolates in seeds. Higher elemental sulphur doses applied as foliar fertilisation (to 64 kgˇha^-1) inco nsiderably increased the sum of alkenyl glucosinolates and decreased the content of indolyl glucosinolates. All that can point to no relationship between the content of glucosinolates in seeds and foliar fertilisation with elemental sulphur dose as in that form only 2% of sulphur is uptaken directly by leaves. The remaining amount together with precipitation reaches soil where it is transformed by microorganisms and gradually included in the plant metabolism [4].

The foliar fertilisation of oilseed rape with ionic sulphur and soil fertilisation with elemental sulphur in the present research increased the content of glucosinolates in seeds proportionally to the dose applied. The highest total content of glucosinolates (12.8 µMˇg^-1 of dry matter), including the sum of alkenyl glucosinolates (7.48 µMˇg^-1 of dry matter), was recorded in seeds of plants when soil was fertilised with elemental sulphur at the dose of 60 kgˇha^-1. A varied level of glucosinolates in seeds affects the pathogen-plant relationship. As shown by Trzciński [17] and Drozdowska et al. [5], the seeds collected from objects where soil was fertilised with elemental sulphur at 60 kgˇha^-1 were least infected by pathogenic fungi.

Sulphur fertilisation significantly increased the total content of alkenyl glucosinolates in seeds, however, its effect on the indolyl glucosinolates was not clear. An increased content of alkenyl glucosinolates in seeds of oilseed rape fertilised with sulphur was mostly due to an increase in the content of progoitrin. Mean results of the three-year experiment show that sulphur enhanced the accumulation of only this form of alkenyl glucosinolate. Similarly Zhao et al. [26] showed that fertilisation with sulphur mainly increased the content of alkenyl glucosinolates. Apparently, an increased dose of sulphur applied stimulates the synthesis of the sulphur-containing aminoacid methionine which is a precursor of all alkenyl glucosinolates present in oilseed rape [25]. With a low supply with sulphur, a decrease in the level of these metabolites can be due to their limited biosynthesis or a decreased content of available methionine [3].

The present research showed that ‘Star’ spring oilseed rape cultivated on soil of a low content of sulphur reacted with the highest increase in yield to foliar fertilisation with elemental sulphur at the dose of 60 kgˇha^-1. The content of glucosinolates in seeds collected from that object (10.8 µMˇg^-1 of dry matter) was comparable with the control (11.9 µMˇg^-1 of dry matter) and with the results reported by COBORU (10.0 µMˇg^-1 of dry matter) [18].

CONCLUSIONS

1. Oilseed rape seed yield and its quality were significantly modified by the sulphur application method and dose as well as the interaction of all the factors studied in the present experiment.

2. The highest mean seed yield was obtained from plants when elemental sulphur was applied in foliar fertilisation at the dose of 60 kg Sˇha^-1. The seeds collected from these objects showed the level of glucosinolates comparable to that of the sulphur non-fertilised object.

3. The soil fertilisation with elemental sulphur at the dose of 60 kgˇha^-1 applied increased the content of the total content of glucosinolates, mainly due to the increase in alkenyl glucosinolates, out of which the content of progoitrin increased most considerably, deteriorating the technological and nutritional seed value.

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Piotr Kachlicki
Institute of Plant Genetics
Polish Academy of Sciences
Strzeszyńska 34, 60-479 Poznań, Poland
e-mail:
szulc@atr.bydgoszcz.pl (Piotr Szulc)
drozd@atr.bydgoszcz.pl (Lucyna Drozdowska)
pkac@igr.poznan.pl (Piotr Kachlicki)

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