Plant Prod. Sci. 13(2): 150―155 (2010)

Glucosinolate Content in Rapeseed in Relation to Suppression of Subsequent Crop

Satoko Yasumoto1, Morio Matsuzaki1, Hisako Hirokane2 and Kensuke Okada3

(1Biomass Production and Processing Research Team, National Agricultural Research Center, 3-1-1, Kannondai, Tsukuba 305-8666, Japan; 2Vegetable and Tea Function Research Team, National Institute of Vegetable and Tea Science, 2769, Kanaya, Shimada 428-8501, Japan; 3Research Strategy Offi ce, Japan International Research Center for Agricultural Sciences, 1-1, Ohwashi, Tsukuba 305-8686, Japan)

Abstract: In crop rotations that include rapeseed ( napus L.), the growth of the crops following rapeseed is sometimes inhibited. The aim of this study was to assess the role of (GSLs) in the inhibitory effect. Three cultivars with zero erucic acid content (Asakano-natane, Kizakino-natane, Nanashikibu: single-low cultivars) and one cultivar with zero erucic acid and low GSL contents (Kirariboshi: double-low cultivar) were grown. The GSL contents differed greatly depending on plant part, stage of development, and cultivar. and gluconapin were detected mainly in the seeds of the single-low cultivars. Their contents either did not change or increased slightly during the reproductive stage. The double-low cultivar Kirariboshi contained almost none of either progoitrin or gluconapin in any part during the reproductive stage. Glucobrassicanapin, glucobrassicin, and were detected, mainly in the roots, of all four cultivars, and tended either to decrease or to remain steady as plants matured. Dense stands of rapeseed seedlings that had germinated from seeds dropped at harvest grew together with the subsequent crop. The GSL contents in the leaves and roots of these seedlings were high. These results suggest that the GSLs in the seeds of single-low cultivars, in the roots of both types at harvest, and in the leaves and roots of volunteer seedlings are the candidate cause of the generally observed phenomenon of inhibited growth of the crop following rapeseed.

Key words: Allelopathy, Gluconasturtiin, Glucosinolates (GSLs), Rapeseed (Brassica napus L.).

Rapeseed (Brassica napus L.) is an important crop for rape mulch, in soil reduced weed seed germination the production of vegetable oil for human consumption, (Peterson et al., 2001). Both volatile and water-soluble and of biodiesel more recently. In warmer regions, such as hydrolysis products of GSLs inhibited seed germination the Yangtze River basin in China, in northern India, and in (Brown and Morra, 1996). Oleszek (1987) reported that central to western Japan, rapeseed is usually grown as a volatiles from B. napus leaves inhibited the germination winter crop in rotation with other summer crops. and the root growth of some weeds and crop plants, but Rapeseed and other Brassica species have phytotoxic effects did not study double-low cultivars or other plant parts. on the subsequent crop (Vera et al., 1987). For example, in Assuming that GSLs were responsible for the suppression Japan, the early growth of sunfl ower and soybean planted of summer crops after rapeseed, we postulated four immediately after rapeseed was severely suppressed, but hypotheses: (i) The GSLs in the leaves of rapeseed that are not that after barley or winter fallow (Okada et al., 2008). usually shed before harvest inhibit the growth of the Cruciferous plants, including rapeseed, contain subsequent crop as the leaves decompose. (ii) The GSLs in glucosinolates (GSLs) in their parenchyma (Röbbelen and the empty pod-shells, stems, and branches cast out by the Thies, 1980). All GSLs are degraded by endogenous combine harvesters, and many seeds left from the shattered (thioglucoside glucohydrolase EC3.2.3.1) pods inhibit the growth of the subsequent crop. (iii) GSLs in (Kawagishi, 1985). The major GSLs in rapeseed and their the rapeseed roots all of which remain in the soil at harvest major breakdown products are listed in Table 1. These affect the growth of the subsequent crop as the roots products inhibited the growth of fungi and oomycetes decompose. (iv) The GSLs in the young plant tissues or living (Smith and Kirkegaard, 2002) and nematodes (Potter et plants (voluntary seedlings) that had sprouted from the al., 1998). In addition, volatilization of seeds left after harvest and emerging together with the (ITC), one of the GLS degradation products from turnip- subsequent crops inhibit the growth of the subsequent crop.

Received 6 October 2008. Accepted 26 October 2009. Corresponding author: S. Yasumoto ([email protected], fax: +81-298-38-7038).

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Table 1. Semi-systematic names, trivial names, and major breakdown products of major glucosinolates in rape. Semi-systematic name Trivial name Major breakdown product Volatility of product 2-Hydroxy-3-butenyl Progoitrin 5-Vinyloxazolidene-2-thione Non-volatile 3-Butenyl glucosinolate Gluconapin 3-Butenyl isothiocyanate Volatile 4-Hydroxy-3-indolylmethyl glucosinolate 4-Hydroxy-Glucobrassicin Unidentifi ed Non-volatile 4-Pentenyl glucosinolate Glucobrassicanapin 4-Pentenyl isothiocyanate Volatile 3-Indolylmethyl glucosinolate Glucobrassicin Indole-3-acetonitrile Non-volatile 2-Phenylethyl glucosinolate Gluconasturtiin Phenylethyl isothiocyanate Volatile Composed from Brown and Morra (1996) and Vierheilig et al. (2000).

Fig. 1. Changes in the contents of major glucosinolates in different parts of rapeseed cv. Kizakino-natane in 2005. Seed was sown on 13 October 2004. Each value is the mean of three or four replicates and line shows standard error.

The purpose of this study was to assess the changes in cultivars), Kirariboshi (Norin No. 48) which has no erucic GSL contents in various parts of rapeseed plants at the acid and only a small amount of GSL in the seed (double- seedling and reproductive stages and to test the above low cultivar). hypotheses. 2. Field experiments Materials and Methods Field experiments were conducted in 2004–2005 and 1. Plant materials 2005–2006 in the Yawara Experimental Field (Tsukubamirai We grew four rapeseed cultivars, Asakano-natane (Norin City, Ibaraki, Japan) of the National Agricultural Research No. 46), Kizakino-natane (Norin No. 47), and Nanashikibu Center (Tsukuba City, Ibaraki, Japan). The experiments (Norin No. 49) which have no erucic acid (single-low were conducted in a rotational paddy field in upland

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Fig. 2. Changes in the contents of major glucosinolates in different parts of rapeseed cv. Kirariboshi in 2005. Seed was sown on 13 October 2004. Each value is the mean of three or four replicates and line shows standard error.

conditions. Seeds were sown on 13 October 2004 and 27 were frozen in liquid nitrogen and then vacuum-freeze- October 2005, in rows 0.3 m apart, with 5 cm plant spacing dried (Laytant LFD-600CS2, Laytant, Kanagawa, Japan). in 30-m×12-m plot. One plot was prepared for one cultivar. Before planting, fertilizer was applied at 7.2 g N m-2, 6.0 g 4. Extraction of GSLs and preparation of desulfo-GSLs -2 -2 P2O5 m , and 6.0 g K2O m . Additional ammonium sulfate GSLs were analyzed as previously described (Ishida et was top-dressed at the beginning of stem elongation and at al., 1995; Kim et al., 2004). Freeze-dried samples were fl owering at 4.0 g N m–2 each time. Weeds were removed ground to a fine powder in a UDY mill (UDY, Tokyo, manually. Japan). A ground sub-sample (200 mg) was then placed in An experiment on rapeseed seedlings that had a 10-mL test tube. Crude GSLs were extracted with 5 mL germinated from seeds dropped from disrupted capsules 80% (v/v) boiling methanol in the capped test tube in a to analyze voluntary seedlings was conducted in July 2006. water-bath at 70ºC for 30 min during which the endogenous myrosinase was inactivated. The mixture was 3. Sampling then centrifuged (21,040 g, 3 min at 4ºC), and the On 25 April (fl owering time), 9 and 23 May, and 6 and resulting supernatant was collected. The residue was re- 20 June (maturing time) 2005, and on 14 and 25 April extracted twice. The bulked crude extract was applied to a (flowering time), 23 May, and 20 June (maturing time) mini-column (using a 1000-μL pipette tip) packed with 2006, three to four plants were collected. On 8 July 2006, DEAE-Sephadex A-25 (40 mg dry weight). GSLs were seedlings at the one- to two-leaf stage were collected from desulfated with 0.5 mL 0.2% aryl sulfatase (15 units). 1-m×1-m quadrat in three replications and we counted the Aqueous sinigrin (allyl glucosinolate) solution (100 μL, 0.2 plants in each sampling area. Immediately after sampling, mg mL–1) was added to the column as an internal all samples were separated into leaves, pods (further standard. After reaction at room temperature overnight, separated into seeds and shells at harvest), stems the desulfo-GSLs were eluted three times into a 10-mL test (hypocotyls in the case of seedlings) and roots. The samples tube with 1 mL distilled water. The extracts were Millipore-

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Table 2. Differences in contents of major glucosinolates in different parts of rapeseed cv. Kizakino-natane and cv. Kirariboshi at harvest. 4-Hydroxy- Gluco- Progoitrin Gluconapin Glucobrassicin Gluconasturtiin total Plant parts Cultivars Glucobrassicin brassicanapin μmol g-1 D.W. 2005 Seeds Kizakino-natane 26.33±2.83** 12.69±1.35** 0.88±0.23 n.d. 0.04±0.01* n.d. 39.94±4.41* Kirariboshi 0.25±0.15 0.15±0.09 1.45±0.06 1.26±0.17** n.d. n.d. 3.11±0.14 Shells Kizakino-natane 2.39±0.11** 0.15±0.01** n.d. n.d. n.d. n.d. 2.54±0.12** Kirariboshi n.d. n.d. n.d. n.d. n.d. n.d. n.d. Stems Kizakino-natane n.d. n.d. n.d. n.d. n.d. n.d. n.d. Kirariboshi n.d. n.d. n.d. n.d. n.d. n.d. n.d. Roots Kizakino-natane 0.31±0.09 0.14±0.02* n.d. n.d. 0.67±0.05 0.38±0.02 1.64±0.16 Kirariboshi n.d. n.d. n.d. 0.34±0.08 1.50±0.47 0.34±0.00 2.19±0.55 2006 Seeds Kizakino-natane 37.88±1.27** 14.94±2.29** 0.67±0.03 n.d. 0.05±0.01 0.10±0.03* 53.01±2.23** Kirariboshi 0.14±0.00 0.06±0.03 0.90±0.27 1.21±0.14** 0.02±0.00 n.d. 1.68±0.09 Shells Kizakino-natane 0.24±0.04* 0.14±0.01** n.d. n.d. n.d. n.d. 0.34±0.09 Kirariboshi n.d. n.d. n.d. n.d. n.d. n.d. n.d. Stems Kizakino-natane trace trace n.d. trace n.d. n.d. 0.03±0.00 Kirariboshi trace trace n.d. n.d. n.d. n.d. 0.02±0.00 Roots Kizakino-natane 0.10±0.04 0.09±0.01* n.d. 0.05±0.01 0.07±0.01 0.32±0.19 0.57±0.32 Kirariboshi n.d. n.d. n.d. 0.01±0.00 0.12±0.02 0.01±0.00 0.14±0.02 Each value is the mean of three or four replicates ± standard error and is adjusted to sinigrin (internal standard). n.d.=not detected. *, **, signifi cant differences at P<0.05, 0.01. respectively.

fi ltered (0.45 μm) and analyzed for GSLs by HPLC. Results 5. HPLC analysis 1. Changes in GSL contents in different plant parts The desulfo-GSLs were separated and detected by the during reproductive stage methods of Ishida et al. (1995) and Kim et al. (2004) with In the single-low cultivar Kizakino-natane, the content of slight modifi cation. HPLC was performed with a class-VP progoitrin in pods was high, and that in other parts was low chromatograph (Shimadzu, Kyoto, Japan) fitted with an and decreased with maturation (Fig. 1). The contents of Intersil ODS-3 column (250 mm×4.6 mm i.d., particle size gluconapin and 4-hydroxy-glucobrassicin in pods increased 5 μm; GL Science, Tokyo, Japan). The temperature of the with maturation; those in other parts were low and column oven was set at 35ºC. The injected sample volume decreased with maturation. Contents of glucobrassicanapin, was 20 μL. GSLs were eluted and separated by linear glucobrassicin, and gluconasturtiin were higher in roots gradient elution with deionized water and acetonitrile as than in other parts and decreased with time. The other described (ISO, 1992) at a fl ow rate of 1.0 mL min–1. The single-low cultivars showed similar results (data not shown). eluates were detected at 228 nm by a UV detector (SPD- In the double-low cultivar Kirariboshi, the contents of 10AVvp; Shimadzu, Kyoto, Japan). Three replications were progoitrin and gluconapin were almost nil in all parts (Fig. used per sample. Each GSL was identifi ed by comparison 2), but 4-hydroxyglucobrassicin in pods increased toward with reported chromatograms (Ishida et al., 1995). harvest and that in other parts were low and decreased with maturation. Some glucobrassicanapin was also detected in 6. Statistical analyses pods at harvest. The roots of Kirariboshi contained almost Results are presented as means ± standard error of the as much glucobrassicanapin, glucobrassicin, and mean. Analysis of variance was performed with the statistics gluconasturtiin as those of Kizakino-natane. program SPSS 11.0J (SPSS Inc., Chicago, IL, USA). The contents of major GSLs in the single-low cultivar were 2. GSL contents at harvest compared with those in the double-low cultivar by t -test at At harvest, all leaves had fallen. In the single-low cultivar the 5% and 1% levels of signifi cance. Kizakino-natane, progoitrin and gluconapin occurred mainly in the seeds. In the double-low cultivar Kirariboshi,

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Fig. 3. Contents of glucobrassicin and gluconasturtiin in roots of rapeseed at harvest (20 June 2005 and 2006). Each value is the mean of three or four replicates and line shows standard error. ■, 2005; □, 2006.

Table 3. Differences in the contents of major glucosinolates in rapeseed cv. Kirariboshi seedlings germinated in the succeeding crop.

4-Hydroxy- Gluco- Total contents of Progoitrin Gluconapin Glucobrassicin Gluconasturtiin Plant parts Glucobrassicin brassicanapin major Glucosinolates mmol m-2 Leaves 12.7±5.4 5.8±2.1 0.4±0.4 620.6±286.9 6.9±3.9 176.2±63.7 819.5±364.2 Hypocotyls 0.7±0.2 0.5±0.1 0.1±0.1 7.2±1.4 0.9±0.1 11.7±1.0 21.0±2.1 Roots 3.8±0.0 1.4±0.6 0.5±0.1 48.7±18.7 12.5±0.3 233.4±70.3 298.2±86.0 Each value is the mean of three replicates ± standard error and is adjusted to sinigrin (internal standard).

the contents were very low (Table 2). Glucobrassicin and gluconasturtiin were present mostly in the roots in both cultivars. At harvest, there were no signifi cant differences (P > 0.05) between single- and double-low cultivars in the contents of these two GSLs in their roots (Table 2, Fig. 3). However, the contents differed with the year (Fig. 3).

3. GSL contents in seedlings In the fi eld (where the preceding crop was the double- low cultivar Kirariboshi), many rapeseed seedlings germinated in early summer. The content of gluconasturtiin in their roots was markedly high (Fig. 4). The contents of glucobrassicanapin in the leaves and roots were also Fig. 4. Contents of glucosinolates in rapeseed seedlings germinated moderately high. We calculated the amount of each GSL from seeds of double-low cv. Kirariboshi. Each value is the mean of three replicates and line shows standard error. ■, Leaves; □, per unit land area (based on an average density of 591 Hypocotyls; and , Roots. plants m–2). The highest was glucobrassicanapin in leaves, followed by gluconasturtiin in roots, gluconasturtiin in leaves, and glucobrassicanapin in roots. Total major GSLs could not be responsible for the inhibition of the growth content in roots was 298 mmol m-2, which corresponded to of the subsequent crop due to GSLs. The GSL content of 26% of that in the whole plant (Table 3). rapeseed leaves at flowering is much lower than that of stems and roots (Rothe et al., 2004). In Brassica juncea L., Discussion the GSL content of leaves reportedly declined rapidly To elucidate the cause of the poor growth of crops after following incorporation into soil, and only small quantities rapeseed, we analyzed the changes in the contents of GSLs remained after 6 d (Gary and Lincoln, 1999). Therefore, in different plant parts. little or no GSLs from the fallen leaves remain at the Although content of glucobrassicanapin of leaves was sowing of the next crop. high in the younger plants (Fig. 4), the GSL contents of At harvest, significant amounts of progoitrin and leaves at fl owering were very low or almost nil (Figs. 1, 2). gluconapin were found in pods in the single-low cultivar Therefore, the fallen leaves of rapeseed after flowering Kizakino-natane (Fig. 1), mostly in the seeds (Table 2). On

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the other hand, the double-low cultivar Kirariboshi Gary, D.B. and Lincoln S.D. 1999. Characterization of volatile contained only small amounts in pods (Fig. 2) or in seeds sulphur-containing compounds produced during decomposition and shells (Table 2). Stems of both cultivars contained of Brassica juncea tissues in soil. Soil Biol. Biochem. 31: 695-703. almost no GSLs, but the roots contained some (Table 2). Höglund, A.-S., M. Lenman, A. Falk and Rask L. 1991. Distribution Thus, GSLs in seeds dropped at harvest of the single-low of myrosinase in rapeseed tissues. Plant Physiol. 95: 213-221. cultivar or in the roots of both cultivars could affect the Ishida, M., Y. Okuyama, Y. Takahata and Kaizuma N. 1995. Varietal diversity of seed glucosinolates content and its composition in growth of the subsequent crop. Japanese winter rape (Brassica napus L.). Breed. Sci. 45: 357-364. The glucobrassicanapin content of leaves and ISO 1992. Rapeseed–determination of glucosinolates content, ISO gluconasturtiin content of leaves and roots in the volunteer 9167-1. International Organization for Standardization, Geneva. rapeseed seedlings were very high: 621, 176, and 233 mmol –2 Kawagishi, S. 1985. Glucosinolates–their enzymatic degradation, and m , respectively (Table 3). Waligora (1996) reported that reactivity and toxicity of degradation products. Nippon Shokuhin rapeseed GSLs reduced seed germination rate and Kogyo Gakkaishi 32: 836-846*. seedling growth of lettuce. Brown and Morra (1996) Kim, S.-J., Jin S., and Ishii G. 2004. Isolation and structural reported that volatiles from intact roots (5.5 g in dry elucidation of 4-(β-D-glucopyranosyldisulfanyl) butyl glucosinolate weight) of rapeseed inhibited the germination of lettuce. from leaves of rocket salad (Eruca sativa L.) and its antioxidative The germination rate was about 30% of control on 8.5-cm activity. Biosci. Biotechnol. Biochem. 68: 2444-2450. ×8.5-cm blotter paper (the area was 72.25 cm2). The major Kirkegaad, J.A., B.J. Smith and Morra M.J. 2001. Biofumigation: soil- GSL in the roots was gluconasturtiin (4.5 μmol g–1 dry wt). borne pest and disease suppression by Brassica roots. Root Res. 10 Therefore, the gluconasturtiin content in the sample was (Extra issue 1): 416-417. 24.75 μmol and the amount of gluconasturtiin per unit Okada, K., M. Matsuzaki and Yasumoto S. 2008. Effect of the area was roughly 3.4 mmol m–2. On the other hand, the preceding crops in rice-based double-cropping systems including observed amount of gluconasturtiin in the roots of rapeseed and sunfl ower. Jpn. J. Crop Sci. 77 (Extra issue 1): 96-97*. seedlings was 233 mmol m–2. These results suggest that the Oleszek, W. 1987. Allelopathic effects of volatiles from some Cruciferae species on lettuce, barnyard grass and wheat growth. GSLs in volunteer rapeseed seedlings affected the growth Plant Soil 102: 271-273. of succeeding crops in rhizosphere. Peterson, J., R. Belz, F. Walker and Hurle K. 2001. Weed suppression by Kirkegaard et al. (2001) reported on the biofumigational release of from turnip-rape mulch. Agron. J. 93: 37-43. functions of GSLs originating from roots. Rumberger and Potter, M.J., K. Davis and Rathjen A.J. 1998. Suppressive impact of Marschner (2004) reported that the rhizosphere bacterial glucosinolates in Brassica vegetative tissues on root lesion community composition was correlated with the GSL nematode Pratylenchus neglectus. J. Chem. Ecol. 24: 67-80. concentrations in roots. Höglund et al. (1991) reported Röbbelen, G and Thies W. 1980. Variation in rapeseed glucosinolates that GSLs in cortical parenchyma cells coexisted with the and breeding for improved meal quality. In S. Tsunoda, K. Hinata enzyme myrosinase or thioglucoside glucohydrolase, and and C. Gomez-Campo eds., Brassica Crops and Wild Allies. Japan proposed that the enzymes had a nonspecifi c defense role. Scientifi c Societies Press, Tokyo, 285-299. Rapeseed seedlings had many small roots and their surface Rothe, R., H. Hartung, G. Marks, H. Bergmann, R. Götz and Scöhne area was large, and this could be one of the reasons for the F. 2004. Glucosinolate contents in vegetative tissues of winter rape high GSL content in the roots of rapeseed seedlings. Our cultivars. J. Appl. Bot. 78: 41-47. results suggest that the GSLs in the roots remaining after Rumberger, A. and Marschner P. 2004. 2-Phenylethylisothiocyanate harvest and in the leaves and roots of volunteer seedlings concentration and bacterial community composition in the are the major cause of the suppression of the growth of the rhizosphere of fi eld-grown canola. Func. Plant Biol. 31: 623-631. subsequent crop. The GSLs in the dropped seeds of single- Smith, B.J. and Kirkegaard J.A. 2002. In vitro inhibition of soil microorganisms by 2-phenylethyl isothiocyanate. Plant Pathol. 51: low cultivars could also contribute to the suppression. 585-593. These inhibitory effects might be controlled by decreasing Vera, C.L., D.I. McGregor and Downey R.K. 1987. Detrimental the harvest losses and by removing root residues from the effects of volunteer Brassica on production of certain cereal and field. The next step is to elucidate how these GSLs oilseed crops. Can. J. Plant Sci. 67: 983-995. suppress the growth of the subsequent crop. Vierheilig, H., R. Bennett, G. Kiddle, M. Kaldorf and Ludwig-Müller Acknowledgments J. 2000. Diffenrences in glucosinolate patterns and arbuscular mycorrhizal status of glucosinolate-containing plant species. New We thank the staff of NARC for their help in data Phytol. 146: 343-352. collection. Waligora, D. 1996. Rape glucosinolates and alfalfa as allelopathic factors for lettuce seed’s germination. J. Plant Prot. Res. References 37: 109-112. Brown, P.D. and Morra, M.J. 1996. Hydrolysis products of glucosinolates in Brassica napus tissues as inhibitors of seed * In Japanese with English title. germination. Plant Soil 181: 307-316.

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