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Ecotype variability in growth and secondary metabolite profile in Moringa oleifera - Impact of sulfur and water availability Nadja Förster, Christian Ulrichs, Monika Schreiner, Nick Arndt, Reinhard Schmidt, and Inga Mewis J. Agric. Food Chem., Just Accepted Manuscript • Publication Date (Web): 18 Feb 2015 Downloaded from http://pubs.acs.org on February 18, 2015
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Journal of Agricultural and Food Chemistry
Ecotype variability in growth and secondary metabolite profile in Moringa oleifera - Impact of sulfur and water availability
Nadja Förster1*, Christian Ulrichs1, Monika Schreiner2a, Nick Arndt2a, Reinhard Schmidt2b, Inga Mewis2a,3 1
Division Urban Plant Ecophysiology, Humboldt-Universität zu Berlin, Lentzeallee 55/57, 14195 Berlin, Germany 2a
Department Quality, 2bDepartment Plant Nutrition, Leibniz-Institute of Vegetable and Ornamental Crops, Theodor-Echtermeyer-Weg 1, 14979 Großbeeren, Germany
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present address: Julius Kühn Institute, Federal Research Centre for Cultivated Plants,
Institute for Ecological Chemistry, Plant Analysis and Stored Product Protection, KöniginLuise-Str. 19, 14195 Berlin, Germany * Telephone +49 (0)30 2093 46430; fax +49 (0)30 2093 46440; e-mail
[email protected] 1
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Abstract
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Moringa oleifera is widely cultivated in plantations in the tropics and subtropics. Previous
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cultivation studies with M. oleifera focused primarily only on leaf yield. In the present study
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the contents of potentially health-promoting secondary metabolites, (glucosinolates, phenolic
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acids, and flavonoids) were also investigated. Six different ecotypes were grown under similar
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environmental conditions to identify phenotypic differences that can be traced back to the
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genotype. The ecotypes TOT4880 (origin USA) and TOT7267 (origin India) were identified
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as having the best growth performance and highest secondary metabolite production, making
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them an ideal health-promoting food crop. Furthermore, optimal cultivation conditions –
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exemplarily on sulfur fertilization and water availability - for achieving high leaf and
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secondary metabolite yields were investigated for M. oleifera. In general, plant biomass and
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height decreased under water deficiency compared to normal cultivation conditions, while the
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glucosinolate content increased. The effects depended to a great extent on the ecotype.
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Keywords
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Biomass production, Ecotypes, Flavonoids, Glucosinolates, Moringa oleifera, Plant height,
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Phenolic acids, Sulfur fertilization, Water limitation
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Introduction
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Moringa oleifera Lam., the main representative of the order Moringa and originally native to
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the sub-Himalayan region, is commonly grown on plantations in Asia and Africa1. All plant
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parts of M. oleifera are edible and contain a multitude of nutrients such as high levels of
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essential amino acids, iron, calcium, and carotenoids2. M. oleifera can be cultivated in
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different ways, but more often the tree is cultivated intensively in plantations to harvest large
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quantities of leaf material1,3-7. Especially in Asia and Africa this tree is cultivated
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commercially5,7-10. Bellostas et al. (2010)10 reported that Moringa leaves rank first among the
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most widely consumed leafy vegetables in Niger. M. oleifera leaves provide a good basis for
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satisfying the population’s nutritional needs. Additionally, M. oleifera is known to be drought
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resistant and is therefore an important source of food during the dry season, especially in the
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African tropics11.
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Besides its high nutritional potential, M. oleifera has been known in Asia and Africa for
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centuries as a traditional remedy. Antimicrobial, anti-inflammatory, detoxifying, and
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anticancerogenic effects of M. oleifera were described among other effects in literature
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(reviewed in 9). These health-promoting effects have largely been attributed to glucosinolates
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or rather their biological active hydrolysis products.
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It is known that M. oleifera ecotypes show a diverse growth performance and leaf mass
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production. M. oleifera leaves are consumed because of their high nutritional value. The high
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levels of potential health-promoting secondary metabolites, especially the glucosinolates,
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have received less attention in cultivation experiments. However, high glucosinolate levels
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might not be present in ecotypes with a high leaf mass productivity. Therefore, in the present
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study ecotypes of M. oleifera with a strong differing growth performance were used to 3
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investigate the possible trade-offs between biomass and glucosinolate accumulation to
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determine the overall best yielding ecotypes. Six ecotypes were used to determine to what
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extent ecotypes show morphological (height, biomass) and chemical (secondary metabolite
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content and composition) phenotypical differences.
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Next to the glucosinolates, M. oleifera leaves also exhibit high contents of phenolic acids and
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flavonoids12-13. Different authors relate health-promoting effects of phenolics to their high
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antioxidative potential (reviewed in
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also analyzed in this study, although the focus remained on the glucosinolates.
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). For this reason, phenolic acids and flavonoids were
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Cultivation conditions can influence plant growth and secondary metabolite contents.
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Different authors have shown in their analyses the influence of fertilization on the growth and
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glucosinolate content of brassicaceous plants15-17. Aromatic glucosinolates represent only a
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small chemical group in glucosinolate containing brassicaceous vegetable leaves and were
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rarely studied in cultivation experiments. One study analyzing the influence of fertilization on
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the leaf glucosinolates of Tropaeolum majus L. showed that sulfur fertilization increased the
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benzyl glucosinolate content, whereas nitrogen fertilization had no significant influence17.
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M. oleifera contained in all plant parts (below-ground and above-ground) very high contents
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of aromatic glucosinolates, which is a characteristic that makes this plant unique. In addition
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to some other trees in the families of Akaniaceae, Bretschneideraceae, or Gyrostemonaceae18,
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M. oleifera has in all plant parts considerable quantities of glucosinolates. Our knowledge of
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the glucosinolate content in tree species is only rudimentary compared to the numerous
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studies that have been conducted on the influence of nitrogen and/or sulfur fertilization on
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brassicaceous leafy plants.
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Several studies have been undertaken about nitrogen and sulfur fertilization and their
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influence on phenolics in Brassicaceae19-22. Li and colleagues20, as well as other different
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authors21-22, found an improving influence of sulfur fertilization on phenolic compounds in
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different Brassica species. Based on the findings of the aforementioned studies, the influence
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of sulfur fertilization on the content and composition of phenolic acids and flavonoids was
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analyzed using M. oleifera ecotypes.
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Due to the fact that one characteristic of M. oleifera is being drought tolerant and harvest take
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place all year round, the influence of water deficiency on the poly-glycosylated aromatic
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glucosinolates was also analyzed. Since M. oleifera is not an annual plant, unlike other
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herbaceous species containing glucosinolate, results are difficult to predict. Analysis of other
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authors showed enhancing or decreasing effects depending on the extent of water stress (e. g.
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23-24
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increasing phenolic content after drought stress27-28, a change in the content and composition
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of phenolics in M. oleifera leaves is conceivable. Therefore, analyses were included in the
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present study.
) and plant species (e. g.
25-26
). Based on different previous studies, which show an
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Since studies on accumulation of secondary metabolites under varying conditions are rare or
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non-existent for M. oleifera, effects of sulfur fertilization and drought stress on plant growth
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and levels of secondary metabolites in the leaves of the different M. oleifera ecotypes were
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investigated. The results will generate information providing insights into an optimal
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cultivation of M. oleifera ecotypes. The overarching aim of the study was to find differences
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in M. oleifera ecotypes in growth and secondary metabolite content to determine the most
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suitable ecotype for leaf and secondary metabolite production. The goal was to identify an
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optimal cultivation variant with the best plant growth performance and the highest secondary 5
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metabolites content. The results provided information about how to achieve a high yield in
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biomass as well as potentially health-promoting ingredients in the cultivation of M. oleifera.
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Materials and Methods
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Cultivation experiments in Großbeeren
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After a pre-screening using 13 different ecotypes of Moringa oleifera Lam., seeds of the six
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best germinating and growing ecotypes were selected for the experiments. The M. oleifera
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ecotypes were pre-cultivated in a greenhouse at the Leibniz-Institute of Vegetable and
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Ornamental Crops in Großbeeren, Germany. Seeds from four ecotypes adapted to different
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climatic conditions were obtained from the AVRDC (“The World Vegetable Center”,
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Taiwan): TOT4880 (origin USA; ecotype 1), TOT5028, TOT7277 (origin Thailand; ecotypes
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2 and 3), TOT7267 (origin India; ecotype 4). Seeds from the other two ecotypes were
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collected in the Philippines and Taiwan (ecotypes 5 and 6). Seedlings (8 weeks old) were
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planted in grid patterns at 0.5 m apart in a 1 m deep enclosed greenhouse bed. Drip irrigation
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and a nebulization system were used to humidify soil and air. In 2010, the mean temperature
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during the vegetation period (from June to October) in the greenhouse was 19.5 °C (2011:
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18.6 °C, 2012: 19.5 °C) and varied during this period between a maximum temperature of
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23.5 °C (in July) and a minimum temperature of 16.3 °C (in October) (2011: 20.5 °C (in
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August) and 14.6 °C (in October), 2012: 22.3 °C (in July) and 15.7 °C (in October)). The
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ventilation temperature was set to 19 °C. Biomass production, height, and total glucosinolate
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content were determined to analyze ecotype differences. Plant samples were taken in August
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2010 (first harvest); November 2010 (second harvest); July 2011 (third harvest); October
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2011 (fourth harvest) and August 2012 (fifth harvest). The leaf/stem ratio of well-established
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plants was analyzed from plant material at the second point of harvest. By this time, the plants
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had grown into completely developed trees. 6
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Furthermore, the influence of different growing variants on the growth and secondary
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metabolite contents (glucosinolates, phenolic acids, and flavonoids) of the six M. oleifera
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ecotypes was analyzed in November 2010 (second point of harvest) and in July 2011 (third
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point of harvest) (normal variant, sulfur variant, water deficiency variant, Suppl. Tab. 1). Per
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variant and ecotype 5 plants were cultivated. Each growing variant was located in a closed
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bed surrounded with concrete, therefore separated from each other. All beds were fertilized
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with 7.04 g/m2 nitrogen (372 g calcium ammonium nitrate per parcel, twice a year), 2.56 g/m2
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phosphorus (Superphospate, 465 g phosphorus pent-oxide per parcel), and 5.76 g/m2
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potassium (Patentkali®, 330 g potassium oxide per parcel) (basic fertilization) once a year at
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the end of April/beginning of May. The plants of the sulfur variant received annually
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0.704 g/m2 sulfur additionally (56 g potassium sulfate per parcel). To reach low to moderate
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water deficiency, in one bed irrigation and the nebulization system (in the whole cabin) was
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switched off in defined periods (August – November 2010, June – September 2011). In the
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cultivation beds containing the normal and sulfur variant a continuous soil water tension of
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approximately 50 – 100 hPa was maintained. In the bed with water deficiency a higher soil
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water tension was determined (second harvest: 380 hPa, third harvest: 175 hPa; Suppl. Fig. 1).
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At the second harvest the upper limit of the field capacity was exceeded (FC ranged between
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63 – 316 hPa, therefore pF = 1.8 - 2.5). The water content of the sandy soil was at this time
