Chilean Journal of Agricultural Research, Vol. 70, No. 2, April-June, 2010, pp. 228-236
Borage (Borago officinalis L.) response to N, P, K,and S fertilization in South Central Chile
Respuesta de borraja (Borago officinalis L.) a la fertilización con N, P, K, y S en el Centro Sur de Chile
Marisol T Berti1, Susana U Fischer1, Rosemarie L Wilckens1, María F Hevia2, Burton L Johnson3
1 Universidad de Concepción, Facultad de Agronomía, Casilla 537, Chillán, Chile
Correspondence Address: Marisol T Berti, Universidad de Concepción, Facultad de Agronomía, Casilla 537, Chillán, Chile, email@example.com
Date of Submission: 24-Sep-2008
Code Number: cj10025
AbstractBorage (Borago officinalis L.) is an oilseed with a high gamma-linolenic acid (GLA) content in its seed. The objective of this study was to determine the response of borage seed yield, oil content, and fatty acid composition to N, P, K, and S fertilizer treatments. Three experiments were conducted in Osorno (40 o 22'S, 73 o 04'W; 72 m.a.s.l.), Chile. The first experiment was conducted during the 2005-2006 growing season, with four N rates (0, 100, 200, and 300 kg N ha -1 ), three P rates (0, 60, and 120 kg P 2 0 5 ha -1 ) and two K rates (0 and 150 kg K 2 0 ha -1 ). The second experiment was conducted in 2006-2007 and evaluated only the effect of four N rates (0, 50, 100 and 150 kg N ha -1 ) was evaluated because there was no response to P and K in the first experiment. The third experiment was conducted in the 2005-2006 and 2006-2007 and evaluated only two rates of S, 0 and 40 kg S ha -1 were evaluated. Seed yield was not affected by N, P, K, or their interactions in any of the experiments. As N rates increased GLA content increased. Mean seed yield increased to 98 kg ha -1 when appliying of 40 kg S ha -1 . Results indicate that borage has a higher response to S applications than N. Further research is needed to determine the interactions between N and S applications, given that the experiments were conducted on soils with high levels of P and K levels.
Keywords: seed yield, gamma-linolenic acid
La borraja (Borago officinalis L.) es una oleaginosa con alto contenido de ácido gamma-linolénico (GLA) en su semilla. El objetivo de este estudio fue determinar la respuesta en rendimiento de semillas, contenido y composición del aceite de borraja, a la fertilización con N, P, K y S. Tres experimentos fueron conducidos en Osorno (40º22 S, 73º04 O; 72 m.s.n.m.), Chile. El primer experimento fue conducido en la temporada 2005-2006, con cuatro dosis de N (0, 100, 200 y 300 kg N ha-1 ), tres dosis de P (0, 60 y 120 kg P2O5 ha-1 ) y dos dosis de K (0 y 150 kg K2O ha-1 ). El segundo experimento fue conducido en el 2006-2007, cuando sólo se evaluaron cuatro dosis de N (0, 50, 100, y 150 kg N ha-1 ) ya que en la primera temporada no se observó respuesta a P y K. El tercer experimento se realizó en 2005-2006 y 2006-2007 y se evaluaron dos dosis de S, 0 y 40 kg S ha-1. El rendimiento de semillas no fue afectado por la dosis de N, P, K o la interacción entre ellos en ninguno de los experimentos. A medida que aumentó la dosis de N se observó un aumento en el contenido de GLA. La fertilización con 40 kg S ha-1 aumentó en promedio el rendimiento de semillas en 98 kg ha-1 . Los resultados indican que la borraja tiene una mejor respuesta a S que a N en las condiciones evaluadas de suelos con alto nivel de P y K; sin embargo, se requiere de un estudio en mayor profundidad para determinar el efecto de las interacciones entre N y S.
Palabras clave: producción de semilla, ácido gama- linolénico.
Borage (Borago officinalis L., Boraginaceae) is an herbaceous annual plant (Janick et al., 1989). Current interest in this crop is for its seed which contains a high content of gamma-linolenic acid (GLA) (all- cis 6, 9, 12-octadecatrienoic acid) in the oil. This acid (GLA) is a precursor of the prostaglandin PGE1 in the human body (Coupland, 2008), which is vital in many body functions, such as antithrombotic inhibitory effects on aggregation of platelets, lowering blood pressure, and inhibiting cholesterol formation (Coupland, 2008). Potential medical uses of GLA include treating atopic eczema to decrease disease symptoms (Horrobin, 2000) and reducing side effects of diabetes, such as vascular damage, altered platelet function, and arteriosclerosis (Coupland, 2008). Commercial seed sources of GLA include evening primrose (Oenothera biennis L.) and some Ribes species. Oil content of borage seeds fluctuates between 300 and 380 g kg -1 of which 20% to 23% is GLA. Oil and GLA content are higher in borage seed compared to those from evening primrose and Ribes spp. (Deng et al., 2001).
Main producers of borage seed are Canada, England, United States, and Chile (Nicholls, 1996). Chile had 2000 ha of borage under contract each year in 2004, 2005, and 2006 becoming an interesting crop alternative for the area. However, contracts were ended in 2007 due to reduction of international prices and cheaper sources of oils containing GLA. The market for borage oil, as for evening primrose oil, fluctuates dramatically with some years of over-supply and others of low production. One of the reasons for this is that the major borage producer is Canada, where growers can produce seed at the lowest cost, but where there is a high risk for crop failure due to early frosts. Also, evening primrose oil from China, another source of GLA, flooded the North American market in 2001 reducing the prices for GLA-containing oils (El Hafid et al., 2002a). Therefore the quantity of borage seed marketed each year is variable, fluctuating between 500 and 2000 t worldwide. Borage seed price fluctuates between US$2.5 to 4 kg -1 of seed depending on supply and seed quality. Seeds higher than 24% GLA are easier to market during years of oversupply. Borage oil price fluctuates between of US$30 to 35 kg -1 in the market place (Lindemann and Merolli, 2006).
Borage is grown usually at higher latitudes to increase GLA content. Most processing companies require a minimum 22% GLA, which is not easy to obtain at latitudes lower than 38º. Southern Chile has climatic and soil conditions favorable for borage seed production (Berti et al., 2002). Borage grows in a broad range of climates and soil pH from 4.3 to 8.5 (Janick et al., 1989); however to obtain a high quality seed oil, the seed development must occur with temperatures below 25ºC.
Borage grows rapidly and it is ready to harvest in about 75 d after sowing. Nutrient requirements for borage are not clear. Previous studies indicate no response to N up to 80 kg N ha -1 in Alberta, Canada, although the lack of response was attributed to high initial soil N content (El Hafid et al., 2002a; 2002b). However, in Egypt, sequential application or foliar N, as urea, increased seed yield and decreased oil content (Refaat et al., 2000). In Australia, borage is fertilized with 500 kg ha -1 of a (13:14:13) N:P:K fertilizer (Laurence, 2004). There are no references on borage requirements of P and S. However, oilseeds, such as canola (Brassica napus L.), require 23 to 34 kg ha -1 of applied S when soil test S are low to medium (Berglund et al., 2007). The total S uptake for canola is 60 kg S ha -1 (Jackson, 2000).
The objective of this study was to determine the response of borage seed yield, oil content and fatty acid composition to N, P, K, and S fertility treatments in southern Chile.
Materials and Methods
Experiments were conducted in Osorno (40º22′ S, 73º04′ W; 72 m.a.s.l.), Los Lagos Region, Chile, in the 2005-2006 and 2006-2007 growing seasons. The experiments were conducted under dryland conditions and no-till. The previous crop was wheat (Triticum aestivum L.). Soil at Osorno is classified as Osorno series (ashy, mesic Typic Haploxerand), and slightly hilly. Climate is classified as cold Mediterranean with rainfall from 1200 to 1500 mm. Average monthly temperature and rainfall at Osorno in the 2005-2006 and 2006-2007 growing seasons, and soil analysis from both seasons are presented on [Table - 1].
Experiment 1. Interaction N, P, and K effect
In the 2005-2006 growing season, the experimental design was a randomized complete block (RCB) with a factorial arrangement (4 x 3 x 2), with four rates of N (0, 100, 200, and 300 kg N ha -1 ), three rates of P (0, 60, and 120 kg P 2 0 5 ha -1 ) and two rates of K (0 and 150 kg K 2 0 ha -1 ) and four replications. The N was applied as urea (46-0-0). Phosphate was applied as triple superphosphate (0-46-0), and K as KCl (0-0-60). Experimental units consisted of six rows, 5 m long and spaced 17.5 cm apart. The experiment was sowed on 31 September 2005 with a seeding rate of 8 kg ha -1 . Glyphosate (N-(phosphonomethyl) glycine; 2 L ha -1 ) was applied 2 d after seeding. At seeding all fertilizers were applied and incorporated in seeding furrows at 2 cm depth, N was not partialized. Plots were hand weeded. The two-center rows of each plot were swathed and 7 d later threshed with a stationary plot combine (according to method developed by Simpson (1993b). Harvest date was 3 January 2006.
Experiment 2. Nitrogen fertility
In the 2006-2007 growing season, only the effect of N was evaluated because there was no response to P and K on most of the characters evaluated the first season. The experiment had four N rates: 0, 50, 100, and 150 kg N ha -1 . The design was a RCB with four replicates and experimental units consisted of six rows, 5 m long and spaced 20 cm apart. Each N rate was split-in-half and hand-broadcasted in two stages: leaf stage (1.2) and rosette stage (2.0) (Simpson, 1993a). Nitrogen fertilizer used was urea. No other fertilizers were applied since the soil analysis indicated soil P and K levels were high. The experiment was planted on 22 August 2006 with a seeding rate of 8 kg ha -1 . Glyphosate (2 L ha -1 ) was applied before seeding. Thereafter plots were hand weeded. The two-center rows of each plot were swathed by hand and 7 d later threshed with a stationary plot combine. Harvest date was 5 January 2007.
Experiment 3. Sulphur fertility
The S fertilizer experiment was conducted in the 2005-2006 and 2006-2007 growing seasons at Osorno. The experimental design was a RCB with two rates 0 and 40 kg S ha -1 and with different split-application treatments and four replications. Treatments were: control treatment (Tl), 25 kg S ha -1 applied at seeding and 15 kg ha -1 applied at stage 2.0 (T2), 15 kg S ha -1 at seeding and 25 kg ha -1 at stage 2.0 (T3), 40 kg S ha -1 at seeding (T4), and 40 kg S ha -1 at stage 2.0 (T5). Planting was done no-till with a preplant application of glyphosate (2 L ha -1 ) for weed control. Seeding dates were 22 September 2005 and 22 August 2006. Seeding rate was 8 kg ha -1 and experimental units had six rows of 5 m long and spaced 20 cm apart.
Harvest was conducted on 3 January 2006 and 5 January 2007 when the first four fruits on main stem started to shed the seed, stage 5(4.0) (Simpson, 1993a; Berti et al., 1998).
In all experiments, plant height was measured at harvest for five plants in each plot. Seed yield was obtained from a 2 m section of both center rows of each plot. Test weight was calculated by determining the weight of 40 mL of seed from a clean seed sample and 1000-seed weight was calculated by counting 250 seeds of each experimental unit.
A sample of 0.1 g of dried ground plant tissue was analyzed by the Kjeldahl procedure to determine total seed N. Seed N evaluations were only conducted for Experiment 1 for the N treatments 0, 100, 200, and 300 kg N ha -1 , and three replications, and for Experiment 3 samples from 2005-2006 for 0 and 40 kg S ha -1 , treatments Tl through T5, and three replications.
Seed oil content was determined on 40 mL of clean dried seeds with a Nuclear Magnetic Resonance (NMR) Analyzer (Newport 4000, Oxford Institute Limited, Oxford, England), at the Department of Plant Sciences, North Dakota State University. Oil content was expressed on a 0% moisture basis. This is the standard procedure for determining oil content of oilseeds (Robertson and Morrison, 1979). Oil yield was calculated multiplying seed yield by oil content.
Fatty acids analysis for Experiment 1 was done only for the samples with 0, 100, 200 and 300 kg N ha -1 , and three replicates. Fatty acid analysis was conducted at the Northern Crops Science Research Laboratory-USDA, Fargo, North Dakota. Seed samples (10 whole borage seeds per sample) were ground with a glass hex wrench on a 2 mL vial. Hexane chloroform sodium methoxide solution (HCSM, 0.5 M, 1 mL) was added to a vial (2 mL). The samples were derivatized by vortexing. Aliquots of the sample (200,uL) were diluted with HCSM (200,μL) in separate vials and injected on to the gas chromatographer (Hewlett-Packard 5890 Series II, Palo Alto, California, USA), equipped with a flame-ionization detector and an autosampler/injector. Analyses were conducted on a DB-23 30 m x 0.25 mm column with a 0.25 μm film (J&W Scientific, Folsom, California, USA). Analysis was conducted as follows: column flow 1.9 mL min-1 with helium head pressure of 200 kPa; injector split flow at 50-100 mL min -1 ; column oven temperature at 190ºC for 5 min followed by a ramp to 220 ºC are 10ºC min -1 with a 1 min hold ending with a ramp to 240 ºC at 20 ºC min -1 with a 5 min hold; and injector and detector temperatures set at 230 and 250ºC, respectively. Standard curves of methyl-caprate, methyl stearate, and methyl oleate from Nu-check 21A and 411 provided standards for calculating equivalent chain length (ECL) values, which were used to make FAME assignments. Each sample was run with two replicates.
Fatty acid composition analysis for 2006-2007 Experiments 2 and 3 was performed at the Facultad de Ingenieria Agricola, Universidad de Concepcion, Chillan, Chile. Gas chromatography (GC) of fatty acid methyl esters (FAME) was performed with a gas chromatograph (Varian 3900, Palo Alto, California, USA) equipped with a flame ionization detector (FID). Analyses were conducted on CP-WAX 52 CB, 30 m x 0.25 mm column with an external diameter of 0.39 mm and the size of filling particle of 0.25 μm. Analysis was conducted with a set temperature of 120 to 240 ºC, in three stages 120 ºC for 3 min, increasing temperature in 3 ºC min -1 , until 210 ºC, maintaining that temperature for 55 min, finally temperatures were increased in 15 ºC min -1 until 240 ºC for 65 min. The temperature detector was set at 300 ºC. The temperature injector was set at 200 ºC, with a flow of 1 mL min -1 . Standard curves of methyl oleate, methyl linoleate, and methyl linolenate were used to confirm response factors for the GC FID that matched those previously reported by Ackman (2002).
Statistical analysis was conducted using standard procedures for a RCBD with a factorial arrangement for Experiment 1, and RCBD for Experiment 2 (Steel and Torrie, 1980). N, P, K, and S effects were considered fixed for all analysis. For Experiment 3, each location-year combination was defined as an `environment′ and was considered a random effect in the statistical analysis. Residual mean squares were compared for homogeneity among environments for each trait. If homogeneous, then a combined ANOVA was performed across environments. Means separation was performed by applying F-protected Least Significant Differences (LSD) comparisons at P ≤ 0.05 level of significance. The estimated variance of pairwise mean differences and the corresponding degrees of freedom were calculated to estimate the correct LSD values for comparison of significant. Combined analysis for Experiment 3 was analyzed as a RCBD for only two treatments 0 and 40 kg S ha -1 and as a RCBD with five treatments, including different split-applications. Linear and quadratic regression models were evaluated for each variable. The regression models and all parameter estimates were significant at P ≤ 0.05. SAS System was used to process the data (SAS Institute, 2005).
Results and Discussion
Experiment 1. Interaction of N, P, and K effect
Plant height. The ANOVA indicated that plant height was affected by N, P, and the interaction between N and K [Table - 2]. The interaction between N and K was significant because when 300 kg N ha -1 and 0 kg K 2 0 ha -1 were applied the plant height was greatly reduced [Figure - 1]; however, this reduction was not significant when 150 kg K 2 0 were added with 0 K added. This occurred because plants branched more when more N was available and this reduced plant height, only when K availability was reduced. Potassium plays an important role in plant turgescence; therefore is more likely to grow in height with higher N if K is available.
Seed yield. Seed yield was not affected by N, P, K, (or their interactions) [Table - 2]. The high soil P and K level explain the lack of response of borage to these nutrients. Other reports indicated also that N fertility levels did not affect seed yield on an experiment conducted at Alberta, Canada, although in this study the non-response to N was explained by the high initial nitrate content in the soils (El-Hafid et al., 2002a; 2002b). Soil nitrate content was 9.3 mg kg -1 for this experiment, classified as a low to medium level (Soil Department, University of Concepcion), which would not explain the non-response to N observed. Seed yields obtained in this study are similar to those obtained by other researchers (El-Hafid et al., 2002a: 2002b). The crop did not have other limitations for growth and development such as water stress, pH or management. Although soil pH is low there was not a concern for high Al which was tested but analysis is not available to report it. Nitrogen fertility experiments conducted in same experimental site and in same or different growing season with other crops such as flax (Linum usitatissimum L.), canola (Brassica napus L.), camelina (Camelina sativa L.), and mustard (Brassica juncea L.) all had a very clear response to increasing N rates. This indicates that there were no other soil limitations to the lack of response, but the nature of borage growth and development. It is hard to determine borage seed yield response to N since the crop shatters heavily. Most of grain yield in borage is shattered which increases the difficulty to measure grain yield response to N. The plant also has an indeterminate growth habit and seed shatter of 75% is common (Berti et al., 2002).
1000-seed weight, test weight and seed N. The 1000- seed weight was affected by N fertility levels, and by the interactions between P x K and the test weight was affected by N fertility levels and the N x K, P x K, and N x P interactions [Table - 2]. Seed weight decreased as N fertility rates were increased from 16.4 g with 0 kg N ha -1 to 12.3 g with 300 kg N ha -1 [Table - 3]. Seed N, and therefore, seed protein content did not increase significantly on this study although there was an increasing trend as N rate increased [Table - 2]. The slightly higher seed N for the treatment with 200 kg N ha -1 may explain the lower 1000-seed weight and test weight at higher N rates. Generally, a higher N fertility increases seed protein content. Protein utilizes less space on the seed than starch, reducing the seed weight and test weight (Otteson et al., 2007). A reduction on thousand-kernel weight as N fertility increased has been reported for spring wheat (Otteson et al., 2007). The interaction between P and K for 1000-seed weight occurred because the seed weight increased from 13.7 g to 17.3 g 1000 seeds -1 when the P fertilizer was increased from 60 to 120 kg P 2 0 5 ha -1 without K fertilizer added, on the other hand, when the same P rates were used and 150 kg K 2 0 ha -1 as added there was not a significant increase in seed weight indicating P is more important than K for seed dry matter accumulation. Both significant interactions, N x P and N x K indicate that highest test weight was observed when rates used were the highest, 42.8 kg hL -1 (300:120 N:P kg ha -1 ) and 39.8 (300:150 N:K kg ha -1 ). The interaction between P x K indicate that lowest test weight was observed with 0 P 2 0 5 kg ha -1 and 150 kg K 2 0 ha -1 .
Oil content and oil yield. Oil content and yield was not affected by N, P, K, or their interactions [Table - 2]. Also, El-Hafid (2002b) did not find effect of N rates on oil and GLA content. In sunflower (Helianthus annuus L.), excessive N fertilization favors vegetative growth and reduces seed oil content (Schemer et al., 2002), effect that was not observed in this experiment. Oil content in borage grown in southern Chile fluctuates between 300 and 330 g kg -1 and this character depends mainly on harvest stage, temperature during seed development, and seed maturity. Oil content increases when the majority of the seed harvested are at or past physiological maturity (Berti et al., 1998).
Fatty acid composition. Nitrogen levels did not have an effect on any of the fatty acids analyzed for this experiment [Table - 2]. Average GLA content was 22.5%. The only other research that has evaluated GLA content for different N rates was El-Hafid et al. (2002b), who did not find significant differences for GLA content. Previous studies in Chile and in North Dakota, USA, have evaluated the factors that influence GLA content and fatty acid composition in borage seeds (Berti et al., 2002). Air temperature during seed development and seed maturity at harvest are the key factors to the fatty acid composition of borage seed oil. Berti et al. (2002) reported GLA content to fluctuate from 18.4% to 26.4% in the northern most and warmer location evaluated, Chillan to Puerto Varas in Southern Chile. The differences in GLA content were associated to cooler temperatures during seed development.
Experiment 2. Nitrogen fertility
Seed yield. This experiment also showed a non significant response of seed yield to N fertility levels as in Experiment 1 [Table - 2] and [Table - 4]. The results were similar to results in Experiment 1, which may be explained for the high shattering of the crop that difficult the ability of measuring differences. As explained for Experiment 1 soil pH, water stress, or other soil limiting condition do not explain the lack of response in this study. The rainfall for this season was much higher than the one before, this may have caused more N to leach in deeper layers of the soil and not be available for borages shallow-root system.
Oil content, yield and fatty acid composition
Nitrogen fertility levels did not have an effect on oil content, oil yield, palmitic acid, stearic acid, oleic acid, and linoleic acid (data not shown) [Table - 4]. The only significant response was observed for GLA [Figure - 2]. As N rates were increased GLA increased; however, the difference between the control treatment and the highest rate was only 0.6 percentage points. Seed oil GLA contents were lower than required by industry (22%). Probably, this was due to high temperatures during seed development. El-Hafid et al. (2002b) did not find significant differences for seed oil GLA with increasing N fertility rates. However, delaying harvest had a significant effect on seed oil GLA due to lower temperatures during seed development. Presumably, a higher N availability allowed the crop to grow vegetative longer, then at harvest the plants from the higher N treatments were at an earlier stage of maturity (5:(1.0)) than the other treatments at probably (5:(4.0)) or more (Berti et al., 2002). Previous research indicates that the first mature seeds from the plants (stage (5:1:0)) are known to have higher GLA content (Berti et al., 1998; 2002). This indicates that the positive effect of N on GLA content may be indirect effect and not an effect of the N itself.
Experiment 3. Effect of sulfur fertilizer
Seed yield. The effect of S application was significant for seed yield when analyzed without split-application effects [Table - 5]. Mean seed yield increased 98 kg ha -1 when 40 kg S ha -1 were applied. There were no significant differences among treatments when all S was applied at seeding or part of it was split at seeding and the rest applied at the rosette stage [Table - 6]. There is no reported information on S requirements for borage. As a reference, S uptake of canola is 60 kg ha -1 and its deficiency decreases seed yield (Jackson, 2000). Canola and wheat are commonly fertilized with 40 kg S ha -1 for soils with low and medium levels in southern Chile where this study was conducted.
1000-seed weight and test weight and seed N. Sulfur rates and the interaction with environment were not significant (P ≤ 0.05) for 1000-seed weight and test weight [Table - 5] and [Table - 7]. Sulfur rates did not have an effect on seed N for the S treatments in the 2005-2006 season [Table - 5]; therefore mean 1000 seed-weight, test weight, and seed N data is not shown.
Seed oil content, yield, and oil composition. Sulfur rates and the interaction with environment were not significant for seed oil content, oil yield, pahnitic acid, estearic acid, oleic acid, and linoleic acid [Table - 5] and [Table - 7]. Sulfur application increased slightly the GLA content. Sulfur application of 22 kg ha -1 increased seed oil content in canola, in sites testing low for S (Jackson, 2000) but this response was not observed in this study.
Seed yield was not affected by N, P, K, or their interactions in the conditions this study was conducted. Both 1000- seed weight and test weight were affected by N fertility levels and some of the interactions between N x K, N x P, and P x K. Seed weight decreased as N fertility rates were increased. As N rates increased GLA content increased significantly by 0.6%. The effect of S application was significant for seed yield. Mean seed yield increased 98 kg ha -1 when 40 kg ha -1 of S was applied. There were no yield differences for the split-applications of S treatments. Results indicate that borage has a response to sulfur applications more than N for the soil studied; further research is needed to determine the interactions between N and S applications.
Funding for this research was provided by FONDEF-CONICYT, project Nº D03-I-1100, Chile. Authors acknowledge the valuable collaboration of technicians and students on plot planting, management and data collection and analysis. Thanks to Wilson Gonzalez, Luis Zaflartu, Alejandro Solis, and Alejandra Villar.
Copyright 2010 - Chilean Journal of Agricultural Research
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