Journal of Applied Botany and Food Quality 88, 1 - 4 (2015), DOI:10.5073/JABFQ.2015.088.001 1Julius Kühn-Institut (JKI), Institute for Resistance Research and Stress Tolerance, OT Groß Lüsewitz, Sanitz, Germany ²Julius Kühn-Institut (JKI), Institute for Resistance Research and Stress Tolerance, Quedlinburg, Germany Effects of growing system and season on the alkaloid content and yield of different sweet L. angustifolius genotypes G. Jansen1, H.-U. Jürgens1, E. Schliephake2, S. Seddig1, F. Ordon2 (Received August 18, 2014) * Corresponding author Summary Nine varieties and two breeding lines of sweet Lupinus angustifolius were cultivated under organic and conventional conditions in North- ern Germany in growing seasons 2010, 2011 and 2012. The alkaloid content was significantly influenced by the growing system and year and also by genotype. The variety ‘Vitabor’ and the breeding line ‘Bo 083521AR’ revealed a very low alkaloid content in all years and cropping systems, while ‘Sanabor’, ‘Borlu’ and ‘Boregine’ had a higher content. In the years 2010-2012 significantly lower alkaloid contents (475 μg g-1) were found under organic conditions than un- der conventional conditions (615 μg g-1). The mean alkaloid level of all varieties and breeding lines in organic farming was highest in 2010 (640 μg g-1) and lowest in 2012 (364 μg g-1), depending on temperature during the seed filling period. Investigations on the yield of L. angustifolius revealed significant effects of the genotype, the year and the growing system. Introduction In the past the use of lupins for feed and food purposes was limited because of their high alkaloid content. Alkaloids are toxic and in particular unlimited feeding of bitter seeds resulted in a fatal disease called lupinosis (HONDELMANN, 1984, 1996). Therefore, the interest in lupins free from alkaloids was steadily increasing (HONDELMANN, 1984). On the one hand this can be achieved by debittering on a large scale and on the other hand by breeding (HONDELMANN, 1996). At the beginning of the 20th century v. Sengbusch (V. SENGBUSCH, 1930) identified some sweet grains of lupins (L. luteus and L. an- gustifolius) which were the starting point for breeding sweet lupins. This opened new possibilities for using sweet lupins because of their high protein content (JANSEN and BALKO, 2012) without quantitative restrictions in feed and as food ingredients due to their functional properties (WÄSCHE et al., 2001). This largely broadened the origi- nal use of bitter genotypes of Lupinus species for soil improvement because of their long taproot (CLEMENTS et al., 1993) and ability to fix nitrogen due to symbiosis with the bacterium Rhizobium (LINDE- MANN and GLOVER, 2003). Due to this the so-called ‘Old World’ species L. albus, L. luteus and L. angustifolius as well the ‘New World’ type L. mutabilis achieved some agricultural importance (HONDELMANN, 1984). Currently in Germany only L. angustifolius is cultivated, mainly due to its re- sistance to anthracnose (Colletotrichum lupini, RUGE-WEHLING et al., 2008). Because of the advantageous features mentioned above, especially because of its ability to nitrogen fixation, L. angustifolius is of special importance in organic farming, in which the use of her- bicides, pesticides, and mineral fertilizers is not allowed. Legume- based crop rotations are an option to supply nitrogen in organic farming systems to enhance soil fertility (MAEDER et al., 2002). In this respect TEMPLE et al. (1994) compared conventional, low input and organic farming concerning yield, weed biomass and percent N in leaves of different crops. For dry beans (legumes) no significant differences were detected between cropping systems in all years. For lupins it is known that the alkaloid content, which is of essential importance for food and feed is influenced by many environmen- tal factors, i.e. differences in fertilizer application (CIESOLKA et al., 2005 and GREMIGNI et al., 2003), soil pH (JANSEN et al., 2012) and ambient temperatures during seed filling (JANSEN et al., 2009). The objective of this study was therefore to assess the effect of (i) the genotype, (ii) the growing system (organic versus conventional farming) and (iii) the year on the alkaloid content and yield of Ger- man L. angustifolius genotypes. Material and methods Plant material and cultivation The seeds of all L. angustifolius cultivars and breeding lines were supplied by the seed company Saatzucht Steinach GmbH and Co. KG, Bocksee, Germany. Field experiments were carried out in four random replications with a plot size of 9.6 m² under organic and conventional farming conditions in Groß Lüsewitz (near Rostock, Mecklenburg-Western Pomerania, Germany, northern latitude: 54.071955, eastern longitude: 12.321031). Determinate types (‘Bo- ruta’ and ‘Haags Blaue’) were sown with a sowing rate of 120 seeds m-2 and branched types (‘Boregine’, ‘Borlu’, ‘Hagena’, ‘Probor’, ‘Sanabor’, ‘Sonate’, ‘Vitabor’, ‘Bo 083517AR’and ‘Bo 083521AR’) with 100 seeds m-2 in March/April in 2010, 2011, 2012 and were har- vested at maturity in July/August of the respective season. The nine varieties and two breeding lines were registered or bred, respective- ly, between 2001 and 2008. Under organic conditions the following crop rotation was applied: potato, spring cereals, legumes, winter cereals, grass clover (twice). No manure, chemical seed treatment and chemical or mechanical weed controlwas conducted. Conven- tional cultivation was done according to good agricultural practice with the same crop rotation. The location Groß Lüsewitz is charac- terized by a loamy sand with a medium soil water holding capacity, pH value of 5.8, long-term mean (1972 - 2007) rainfall of 686 mm and mean temperature of 8.3 °C. Meteorological data for the years 2010 - 2012 were supplied by the University of Rostock, Faculty of Agriculture and Environmental Science, Institute for Environmental Engineering, Field of Hydrology. Sample Collection Yields were estimated at a moisture content of 11 % for each repli- cation separately. In a next step seeds of each replication per geno- type were mixed and pre-crushed using a Laboratory Break Mill SM 3 Brabender® (Brabender® GmbH & Co. KG, Duisburg, Germany) and after that ground to pass a 0.8 mm sieve with a Falling Number laboratory Mill 3100 (Perten Instruments, Hägersten, Sweden). All whole meal samples were stored at 20 °C until analysis. Analysis of alkaloids The alkaloids in whole meal samples were analyzed by an Agilent 7890A gas chromatograph equipped with a flame ionization de- tector (FID) on Agilent J&W Ultra 1 column ( 25 m, 0.20 mm ID, 2 G. Jansen, H.-U. Jürgens, E. Schliephake, S. Seddig, F. Ordon 0.33 μm film thickness) and identified in the mass spectrometer Agi- lent 5975C according to WINK et al. (1995) and BERMUDES TORRES et al. (2002). This method was described in detail by JANSEN et al. (2009). Analyses were carried out in duplicate with variation coeffi- cient lower than 4 %. The alkaloid content was calculated as the sum of the main alkaloids which are angustifoline, isolupanine, lupanine, and 13-hydroxylupanine in narrow leafed lupins. Statistical Analysis The statistical analyses were conducted using the software package SAS (version 9.3) for windows programs (SAS Institute, Inc., Cary, NC, USA). A generalized linear model for the analysis of variance (ANOVA) was applied, using the GLM procedure and a Tukey test (α = 0.05) for comparing means (genotype, year and cultivation sys- tem). Results The field experiments with eleven sweet L. angustifolius over a pe- riod of three years showed significant differences in the alkaloid con- tent between genotype and year. The influence of the growing system was significant, too (Tab. 1), but no significant interaction between genotype and growing system was found. Tab. 1: Results of the ANOVA for alkaloid content. Source Degrees p-value (2010-2012) of freedom Genotype 10 <.0001 Year 2 <.0001 Growing system 1 0.0012 Genotype*Growing system 10 0.9185 The alkaloid content of different genotypes, years and cultivation systems is listed in Tab. 2. The variety ‘Vitabor’ and the breeding line ‘Bo 083521 AR’ showed the lowest alkaloid content in all years and growing systems. ‘Vita- bor’ varied between 69 μg g-1 and 158 μg g-1 in organic cultivation and between 127 μg g-1 and 341 μg g-1 in conventional farming. The breeding line ‘Bo 083521 AR’ varied between 71 μg g-1 and 151 μg g-1 and between 127 μg g-1 and 225 μg g-1, respectively. ‘Boregine’, a well known variety with high yield showed high alka- loid contents between 308 μg g-1 and 1108 μg g-1 in organic systems and between 445 μg g-1 and 1312 μg g-1 under conventional condi- tions. The varieties ‘Sanabor’ and ‘Borlu’ were characterized by a very high alkaloid content in seeds, comparable to the content in seeds of ‘Boregine’. Fig. 1 and Tab. 2 show that higher alkaloid contents were detected in the conventional growing system. Fig. 1: Mean alkaloid content of eleven lupin genotypes cultivated under organic and conventional conditions (LS-means with the same letter are not significantly different). The year and the associated different mean temperature during the growing season had an effect on the alkaloid content of the inves- tigated sweet narrow-leaved lupins. The investigated eleven lupins accumulated a higher amount of alkaloids at higher temperatures (Fig. 2). In the growing seasons 2010 - 2012 all genotypes, with the exception of ‘Haagena’, showed significant differences in yield between the growing systems, i.e. conventional and organic farming (Fig. 3). The highest yield, both in organic and conventional farming was ob- tained by the cultivar ‘Sonate’. In general the yield of the lupins cultivated under organic conditions over the three years was significantly lower (2.96 to 4.02 t ha-1) com- pared to the conventionally cultivated lupins (3.96 to 4.56 t ha-1). Tab. 2: Alkaloid content [μg g-1] of nine varieties and two breeding lines of organic (O) and conventional (C) cultivated sweet narrow-leafed lupins in three years. Genotype Cultivation Year 2010 Cultivation Year 2011 Cultivation Year 2012 Mean value 2010-2012 O C O C O C O C Sanabor 942 1204 572 846 753 777 756 942 Borlu 1058 1273 489 751 508 667 685 897 Boregine 1108 1312 599 1027 308 445 672 928 Haagena 864 1075 421 796 653 557 646 809 Sonate 774 696 726 720 482 578 661 665 Bo 083517 AR 655 833 438 733 283 435 459 667 Probor 707 838 472 792 266 527 482 719 Haags Blaue 398 410 322 448 375 307 365 388 Boruta 220 189 455 594 209 286 295 356 Bo 083521 AR 151 187 71 225 94 127 105 180 Vitabor 158 341 82 170 69 127 103 213 Variability of yield and alkaloid content in L. angustifolius 3 The results of ANOVA for yield of lupins cultivated under conven- tional and organic conditions are given in Table 3. Significant effects of the genotype, the year, and the cropping system were observed as well as respective significant interactions. (Tab. 1), by the analyses of 11 genotypes of sweet narrow leaf lupins during 3 years. Several studies compared quality parameters and the effects of or- ganic and conventional growing systems, but in general only small and partially inconsistent differences were found. Possible effects on human health of organic and conventional food are examined in many studies, e.g. by BRAND and MØLGAARD (2001) as well WINTER and DAVIS (2006). In general only small and partially inconsistent differences between organically and conventionally produced foods were detected. Only for nitrate and vitamin C a tendency to lower and higher contents, respectively was detected under organic condi- tions (BRAND and MØLGAARD, 2001). Nevertheless, further investigations on nutritional important plant contents will be necessary to provide more answers to the question, whether organic or conventional cultivation can enhance the nutri- tional value. Results of our study show that under organic conditions all L. an- gustifolius genotypes exhibited significantly lower alkaloid contents than under conventional conditions in all years. The reason for this may be that a higher N-content in the soil leads to higher concen- trations of alkaloids. GHOLAMHOSSEINPOUR et al. (2011) stated that nitrogen is a constituent of the alkaloids which plays an important role in the synthesis of these ingredients. Therefore, increasing ni- trogen supply leads to an increase in the alkaloid content. This has been shown for many species, e.g. lupins (Lupinus succulentus, JOHNSON et al., 1987), datura (Datura innoxia Mill., AL-HUMAID, 2004), periwinkle (Catharanthus roseus L., GHOLAMHOSSEINPOUR et al., 2011) and for the total glycoalkaloid content in potatoes (MONDY and MUNSHI, 1990). However, also contradictory results are reported e.g. for datura (RUMINSKA and GAMAL, 1978) and potatoes (HAMOUZ et al., 2005). RUMINSKA and GAMAL (1978) have shown that there is no remark- able influence of nitrogen fertilizer on the alkaloid content in Datura innoxia Mill. HAMOUZ et al. (2005) found, that growing under or- ganic conditions did not influence the glycoalkaloid content of po- tatoes. Due to these contradicting results new investigations with respect to the alkaloid content in relation to the growing system, i.e. organic versus conventional were conducted. In 2012 the NH4-N-level at Groß Lüsewitz was equal in soil samples of both cropping systems (0.1 mg 100g-1, 0-30 cm), but the NO3-N- concentration in soil samples derived from conventional cultivation reached a level of 0.3 mg 100g-1 compared to organic soil samples with only 0.1 mg 100g-1. It is known that the alkaloid content is influenced by a lot of environ- mental factors, such as temperature. Higher ambient temperatures during the seed filling led to a higher content of alkaloids (JANSEN et al., 2009) in seeds of 6 older varieties of L. angustifolius regist- ered between 1998 and 2004. As expected, in the present study the different temperatures during the growing period from 2010 to 2012 also had a significant influence on the alkaloid content (Fig. 3). Fur- thermore, genotypic differences were also observed in older studies conducted under organic conditions (JANSEN et al., 2005) only. As already mentioned the threshold for the alkaloid content for ani- mal nutrition is 0.05 % and for human nutrition 0.02 % (JANSEN et al., 2009) in Germany, while it is 200 mg kg-1 for contaminants and natural toxicants in Australia and New Zealand (ABBOTT et al., 2003). Alkaloid contents above this level were also detected by MUQUIZ et al. (1994) in genotypes of sweet Lupinus albus L. from different countries and locations. REINHARD et al. (2006) detected up to 2120 μg g-1 in seeds and flour of “sweet” L. angustifolius geno- types. Small, but significant differences in yield between organic (3.53 t ha-1) and conventional (4.25 t ha-1) growing systems were found for 9 varieties and 2 new breeding lines. Similar results were obtained Fig. 3: Mean yield of L. angustifolius genotypes over a period of three years in organic and conventional growing systems cultivated lupins (all varieties had significantly different LS-means-values, with the exception of ‘Haagena’). Fig. 2: Relationship between mean alkaloid content of eleven lupin geno- types cultivated under organic conditions and mean temperature from flowering to pod ripening (Pearson correlation coefficient R = 0.9997, p = ≤ 0.05). Tab. 3: Results of the ANOVA for yield. Source Degrees F-Statistic Pr > F of freedom Growing system 1 244.65 <.0001 Genotype 10 8.47 <.0001 Year 2 230.47 <.0001 Genotype*Year 20 5.87 <.0001 Genotype*Growing system 10 4.06 <.0001 Genotype*Growing system*Year 20 4.13 <.0001 Discussion Differences in the alkaloid content of sweet L. angustifolius are well known. In the descriptive variety list published by the Federal Plant Variety Office (Bundessortenamt, 2011) the variety ‘Vitabor’ is characterized by a very low alkaloid content, compared to the other tested varieties. This is also confirmed by the results of our work 4 G. Jansen, H.-U. Jürgens, E. Schliephake, S. Seddig, F. Ordon in four years field trials for Pisum sativum and Vicia faba and in one year field trials of L. angustifolius (JANSEN and SEDDIG, 2007). In conclusion the alkaloid content and yield of different narrow- leafed lupins is significantly influenced by the genotype, the growing season and the growing system (organic vs. conventional). Significantly lower alkaloid contents and lower yields were found in organic farming compared to conventional farming. However, due to the lower alkaloid content and the only slightly reduced yield, which holds especially true for the cultivar ‘Haagena’, L. angustifolius is well suited for organic farming. Acknowledgements The Authors thank C. Leesch, M. Jugert and B. Lembke for excellent technical assistance. References ABBOTT, P., BAINES, J., FOX, P., GRAF, L., KELLY, L., STANLEY, G., TOMASKA, L., 2003: Review of the regulations for contaminants and natural toxicants. Food Control 14, 383-389. 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WINK, M., MEISSNER, C., WITTE, L., 1995: Patterns of Quinolizidine Alka- loids in 56 Species of the Genus Lupinus. Phytochem. 38, 139-153. WINTER, C.K., DAVIS, S.F., 2006: Organic foods. J. Food Sci. 71 (9), 117- 124. Address of the authors: G. Jansen, H.-U. Jürgens, S. Seddig, Julius Kühn-Institut (JKI), Institute for Resistance Research and Stress Tolerance, OT Groß Lüsewitz, Rudolf- Schick-Platz 3, 18190 Sanitz, Germany E-Mail: gisela.jansen@jki.bund.de E. Schliephake, F. Ordon, Julius Kühn-Institut (JKI), Institute for Resistance Research and Stress Tolerance, Erwin-Baur-Straße 27, 06484 Quedlinburg, Germany