Fruit yield, essential oil concentration and composition of three anise cultivars (Pimpinella anisum L.) in relation to sowing date, sowing rate and locations

Industrial Crops and Products 42 (2013) 489–499

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Fruit yield, essential oil concentration and composition of three anise cultivars (Pimpinella anisum L.) in relation to sowing date, sowing rate and locations Habib Ullah a,b,∗ , Bernd Honermeier a a b

Institute of Agronomy and Plant Breeding I, Justus Liebig University, Ludwigstrasse. 23, D-35390 Giessen, Germany Institute of Horticultural Sciences, University Agriculture Faisalabad, Pakistan

a r t i c l e

i n f o

Article history: Received 16 December 2011 Received in revised form 20 May 2012 Accepted 7 June 2012 Keywords: Pimpinella anisum Cultivar Sowing date Plant density Fruit yield Essential oil trans-anethole

a b s t r a c t Three field experiments were conducted to evaluate the effect of sowing date and plant density on phenology, fruit yield and essential oil quality of three anise cultivars (Pimpinella anisum L.) at two different ecological conditions Gross-Gerau and Giessen, Germany. The experiments were designed as RCBD under factorial plot arrangement with two factors: cultivars in main plot and plant densities in sub plots realized in two sowing dates. Results showed that aniseed sown on April 1st had higher fruit yield in comparison with two weeks delayed sowing. Delayed sowing induced strong effects on yield contributing parameters such as branches/plant, umbels/plant, fruits/plant and 1000-fruit weight which were all reduced. The highest fruit yield was related to lower plant densities of 39 plants m−2 (Giessen) and 200 and 374 plants m−2 (Gross-Gerau). Higher plant density reduced branches/plant, umbel/plant, fruits/plant and 1000-fruit weight and led to lower fruit yield. The essential oil concentration of anise fruits was not affected by different levels of plant densities. A higher concentration of essential oil was synthesized in delayed sowing date in Gross-Gerau 2009 as compared to earlier sown anise plants. The essential oil concentration of anise showed no remarkable variation regarding sowing date and plant density. Cv. Hild Samen synthesized a significant higher concentration of estragol and trans-anethole. From all tested cultivars cv. Hild Samen was characterized by lowest fruit yield as well as lowest essential oil concentration. For successful cultivation, aniseed should be sown as soon as possible in April with a plant density of 50–200 plants/m2 considering soil conditions. © 2012 Elsevier B.V. All rights reserved.

1. Introduction Anise (Pimpinella anisum L.), which belongs to the family Apiaceae, is an important spice and medicinal plant used for pharmaceutics, perfumery and food industry. The fruits as well as the essential oils are characterized by antispasmodic, antioxidant, antimicrobial, insecticidal, and antifungal effects (Tunc and Sahinkaya, 1998; Gülcin et al., 2003; Özcan and Chalchat, 2006; Tepe et al., 2006; Tirapelli et al., 2007). Its fruits which are called aniseed contain around 1.5–5.0% essential oil mainly composed of volatile phenylpropanoids like trans-anethole with around 90% (Tabanca et al., 2005). In addition, the essential oil of the anise fruit also contains a small proportion of estragol, anisaldehyde, ␥himachalene and cis-anethole (Lawrence, 1984; Askari et al., 1998; Omidbaigi et al., 2003; Rodrigues et al., 2003; Tabanca et al., 2006). Because anise favors warm climatic conditions throughout the growing season it is cultivated primarily in subtropical regions (Reineccius, 1994; Hänsel et al., 1999). Seed yield and essential oil

∗ Corresponding author. Tel.: +49 6419937442; fax: +49 6419937449. E-mail address: [email protected] (H. Ullah). 0926-6690/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.indcrop.2012.06.011

contents may vary considerably depending on various ecological conditions including air temperature, UV radiation, precipitation and soil fertility (Farooqi et al., 1999; Dudai et al., 1992; Figueiredo et al., 2008). The most important factors affecting plant growth and the production of secondary metabolites of anise are sowing date, plant density, water supply and harvesting time studied under field or green house conditions (Maheshwari et al., 1989; Zehtab-Salmasi et al., 2001; Omidbaigi et al., 2003; Awad et al., 2005; Tuncturk and Yildirim, 2006). Because of its sensitivity to low temperatures, the sowing of anise in Germany cannot be carried out in early spring. On the other hand delayed sowing under warmer conditions in spring may lead to shortening the growing cycle, which decreases the amount of UV radiation intercepted by the crops and may reduce the formation of reproductive organs. Additionally, the infection of fungal pathogens like Cercospora malkoffii may be affected by plant development under humid climate conditions. More knowledge about the relationships between sowing date and plant density on the one hand and seed yield or essential oil production on the other hand is needed for optimal cultivation of anise in Germany. For this reason field experiments were carried out to determine the effect of different sowing dates, and planting densities on fruit yield and fruit

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Fig. 1. Monthly average air temperature (◦ C) and monthly sum of precipitation (mm) for the growing seasons 2008 and 2009 at two different experimental stations.

quality of three anise cultivars under different ecological conditions (Fig. 1).

2. Materials and methods 2.1. Experiment design Field experiments were carried out at the research stations Gross-Gerau (GG) (49◦ 45 N and 8◦ 29 E, 90.7 m above sea level, sandy soil, pH 6.4, mean air temperature: 9.4 ◦ C, mean precipitation: 590 mm/year) during 2008–2009 and Giessen (Gi) (50◦ 47 N and 8◦ 61 E, 158 m above sea level, silty clay soil, pH 7.4, mean air temperature: 8.5 ◦ C, mean precipitation: 660 mm/year) in 2009. The experiments were designed with two factors: Cultivars (main plot, 1. Enza Zaden, 2. Pharmasaat, 3. Hild Samen) and sowing rate (sub plot, 1 = 8 g/10 m2 , 2 = 15 g/10 m2 , 3 = 30 g/10 m2 ). Each year fresh seeds of anise cultivars were purchased from seed companies of Enza Zaden GmbH, Pharmasaat GmbH, Hild Samen GmbH and Ari-Saaten GmbH, Germany and used for experimentation. Cultivar Hild Samen was more susceptible to fungal infection and was replaced by cv. Agri Saaten in 2009. The experiments were designed as randomized complete blocks (RCBD) with four replications. All three experiments were combined with two sowing dates (early sowing date and two to three weeks delayed sowing). The plot area was 10 m2 , eight rows with row spacing of 18.7 cm. Before seeding, potassium and phosphorus fertilization was done in accordance with soil analysis. The nitrogen content (NO3 -N, NH4 -N) in the soil (0–90 cm) was 52 kg/ha (Gross Gerau 2008), 36 kg/ha (Gross Gerau 2009) and 43 kg/ha (Giessen 2009). Two sowing dates were conducted on April 1st, April 17 and April 1st and April 15 during 2008 and 2009 respectively at GG station. Anise was sown on April 1st and April 20 at experimental station Gi in 2009. Field experiments were also conducted in 2008 at Gi station and data collected analyzed by GC–MS for identification but not presented due to large variation in fruit yield. After emergence, of anise 40 kg N/ha (NH4 NO3 + CaCO3 ) were applied. Weed control was carried out by application of the herbicide Bandur (Aclonifen) with 3 l/ha as well as by hand weeding. In Gross Gerau anise plants were irrigated three times 10 mm (12.05.2008), 20 mm (27.05.2008) and 20 mm (09.07.2008) in 2008 and two times 20 mm (27.05.2009) and 20 mm (03.06.2009) in 2009 according to irrigation requirements. The harvest was made by a combine harvester at the time of full maturity of the fruits. The fungal disease severity on anise plants was recorded for each plot by grading 1–9, 1: without infection, 9: whole plants are infected.

2.2. Morphological and yield components For morphological analysis, samples of anise plants were taken from two rows (2 m length) in middle of the plot 3–4 days before harvesting. With these plant samples the following yield components were determined: number of primary branches/plant, number of umbels/plant, number of fruits/plant, and 1000 fruit weight (TFW). Prior to harvesting plant height was measured with a yardstick. Dry matter percentage of anise was determined after drying at 40 ◦ C for two days. 2.3. Essential oil extraction Fruit samples (7 g) of each plot were hydro-distilled for 2 h using a Clevenger-type apparatus according to the European Pharmacopoeia (2000). The essential oil content was gravimetrically quantified. Each sample was analyzed two times and the average of both was used for further statistic evaluation. The obtained essential oil was kept at 4 ◦ C until further lab analyses. 2.4. GC and GC–MS analyses The components of the essential oil were analyzed by GCFID (Varian CP 3800), capillary column DB-5 (30 m × 0.25 mm and 0.25 ␮m coating thickness). Helium was employed as the carrier gas with a flow rate of 1.1 ml/min. The temperature was programmed from 60 ◦ C (5 min) to 250 ◦ C with a ramp rate of 5 ◦ C/min and a final hold time of 10 min. Injector and detector were maintained at 260 and 280 ◦ C, respectively. The sample (1 ␮l) was injected with 1:50 split ratio by an autosampler (Varian 8200 CX). The percentage of individual components was computed from peak areas. Response factors of detector and FID normalization were considered for data processing. The identification of the components was carried out by GC–MS (Varian 3900 GC, Varian Saturn 2100 T ion trap mass detector), capillary column VF-5ms (30 m × 0.25 mm, 0.25 ␮m coating thickness). Helium was used as a carrier with a flow rate of 1.1 ml/min. Ionization was realized by electron impact at 70 eV, electron multiplier 2200 V, ion source temperature 230 ◦ C and transfer line temperature 240 ◦ C. Mass spectral data were acquired in the scan mode in the m/z range 35–450. The identification of components based on comparison of Kovat’s retention indices and mass spectra in corresponding data libraries (Adams, 1995; Özcan and Chalchat, 2006) and mass spectra libraries (Weily 90 and NIST 98). Kovat’s retentation indices were calculated from the GC by linear interpolation between bracketing n-alkanes (C8–C24; Alfa Aesar Karlsruhe, Germany). The most important components trans-anethole and

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Table 1 Developmental phases (in days) and sum of air temperature (◦ C) in field experiments with anise, 2008 and 2009. Phases

GG 2008 SD1

Developmental phases (in days) Sowing-germination Germination-flowering Flowering-maturity Germination-maturity Air temperature (sum ◦ C) Sowing-germination Germination-flowering Flowering-maturity Germination-maturity

GG 2009 SD2

SD1

Gi 2009 SD2

SD1

SD2

24 60 58 120

17 53 62 115

19 66 58 123

14 63 59 121

33 64 52 116

28 51 58 128

193 1014 1138 2130

206 948 1178 2106

298 1018 1188 2325

197 1032 1198 2208

439 998 986 1966

369 833 1375 2191

GG, Gross-Gerau; Gi, Giessen; SD1, first sowing date; SD2, second sowing date (2–3 weeks after SD1).

estragol were further identified by co-injection of authentic standards (Roth, Karlsruhe, Germany). 2.5. Statistical analyses Statistical analysis of the data was carried out by using the statistical program PIAF Stat for checking the significance of the different treatments, whereas LSD at the level of 5% probability was used to compare the differences between the means. Correlation analysis was performed by PASW (version 18) to determine the relationship among the characters according to Pearson and Spearman’s rho methods. 3. Results 3.1. Field experiment Gross-Gerau 2008 Anise was sown on April 1st (1st sowing) and April 17th (2nd sowing) which resulted in differences of plant development. The seedlings emerged 24 and 17 days after sowing for the corresponding treatments (Table 1). Flowering began after 60 and 53 days in early and delayed sowing date respectively. The time span between beginning of flowering and full maturation of fruits was 58 days for early sowing and 62 days for delayed sowing time. Plants were harvested 142 and 132 days after sowing in first and second sowing date. Anise plants were infected by the fungal pathogen C. malkoffii in a range from 2.4 to 7.6 (level) which led to brown colored leaf spots. Higher fungal infection was observed in early sowing date at narrow planting densities (until: 7.6 level). The applied sowing rates led to different planting densities of 189, 273 and 315 plants m−2 in early and 200, 292 and 324 plants m−2 in delayed sowing time respectively (Table 2). Seeds of cv. Hild Samen had lower germination rates in both sowing times. In early sowing date, fruit yield of anise was affected by both the cultivar and plant density (Table 2). Cv. Hild Samen had the lowest fruit yield caused by the lowest plant density of 117 plants m−2 . However, the maximal plant density of 315 plants m−2 also led to significantly lower fruit yield in early sowing date, whereas in 2nd sowing date no plant density effect was observed. Significant differences were observed regarding the number of primary branches per plant and the number of umbels per plant between used cultivars and planting densities. Under early sowing date plants with the lowest plant density of 189 plants m−2 had the highest number of primary branches and umbels per plant (Table 2). Anise plants formatted 4.4 primary branches/plant in early sowing date which was reduced to 3.5 branches/plant by delayed sowing (Table 2). Cv. Hild Samen produced a significantly higher number of primary branches and umbels as compared to other cultivars in both sowing dates.

An increased plant density induced lower numbers of primary branches and umbels of anise plants in both sowing dates, but in the second sowing time an interaction regarding primary branches/plant and fruits/plant between plant density and cultivar has to be considered (Table 2). Fruit numbers per plant noticed non-significant results regarding planting densities in early sowing date. In delayed sowing date fruit number per plant was significantly affected by different planting densities as well as by cultivars. In both sowing dates plant stands with the lowest density of 189 plants m−2 and 200 plants m−2 had the highest number of fruits/plant with 101 and 118 fruits respectively (Table 2). There was an interaction between cultivars and plant density with respect to fruit number per plant in the second sowing date. 1000-fruit weight (TFW) of anise was at a level of about 2.30–2.51 g (Table 2). Only small differences between the treatments could be found. TFW was not affected by the treatments in early sowing date. On the other hand delayed sowing date induced a significant difference between the tested cultivars. Within all treatments essential oil concentration of aniseed reached a maximum level of 2.90% (2.30–2.90%). In early sowing date there was an interaction between cultivar and plant density (Table 3). A significant lower amount of essential oil was synthesized by cv. Hild Samen as compared to other cultivars. Essential oil yield (EOY) was significantly influenced by plant density and cultivar in first sowing date but only by cultivar in the second sowing date (Table 3). Lower planting densities led to higher EOY in the first sowing which was related to higher fruit yields. The compounds trans-anethole and estragol were not statistically affected by varying planting densities in both early and delayed sowing dates (Table 3). The trans-anethole content in the essential oil ranged from 92 to 97%. Contrary to that the compound estragol was at a very low level of around 0.5 to 1.0% of total essential oil (Table 3). Essential oil content was positively and significantly associated with fruit yield (r = 0.667). Fruit yield and essential oil concentration showed negative correlations with trans-anethole (r = −0.745) and (r = −0.691) respectively in early sowing date (Table 4). Cv. Hild Samen led to significant higher concentrations of estragol and trans-anethole 1.07, 0.97% and 96.9, 96.2%, respectively in early and delayed sowing dates (Table 3).

3.2. Field experiment Gross-Gerau 2009 In 2009 anise seedlings emerged 19 and 14 days after sowing for the corresponding sowing dates (Table 1). Flowering began 66 and 63 days after emergence for first and second sowing respectively. Time span between beginning of flowering and full maturation of fruits was 58 and 59, respectively. Anise plants were harvested after 142 (1st sowing) and 134 (2nd sowing) days. A similar increased

492 Table 2 Effect of different cultivars (CV) and planting densities (PD) on plant height (PH) (cm), number of primary branches per plant (PB), number of umbels per plant (UN), number of fruits per plant (FN), 1000-fruit weight (TFW) (g) and fruit yield (FY) (dt/ha at 91% DM) of anise at early and delayed sowing date in Gross-Gerau 2008. CV

PD

Gross-Gerau 2008 1st sowing date (1.04.2008)

PD CV PD × CV

PH (cm)

PB (No.)

UN (No.)

FN (No.)

TFW (g)

FY (dt/ha)

Pl. (m−2 )

PH (cm)

PB (No.)

UN (No.)

FN (No.)

TFW (g)

FY (dt/ha)

189 273 315 309 350 117

44 43 42 44 43 42

4.4 3.7 2.8 3.2 3.1 4.6

5.9 4.8 4.4 4.2 4.2 6.7

101 87 95 62 68 152

2.49 2.38 2.38 2.40 2.40 2.50

4.5 3.7 3.4 4.5 4.6 2.4

200 292 324 336 354 126

41 41 41 42 41 40

3.5 2.9 2.4 2.6 2.6 3.7

5.0 4.0 3.7 3.6 3.7 5.4

118 89 70 77 86 115

2.39 2.33 2.40 2.30 2.32 2.51

5.1 4.9 4.2 5.5 5.4 3.4

ns ns ns

0.8 0.8 ns

1.2 1.2 ns

ns 69 ns

ns ns ns

0.74 0.74 ns

ns ns ns

1.0 1.0 1.7

ns 2 ns

38 38 66

ns 0.14 ns

ns 1.1 ns

LSD at 5% level: CV 1, Enza Zaden; CV 2, Pharmasaat; CV 3, Hild Samen; PD: 1 = 8 g/10 m2 , 2 = 15 g/10 m2 , 3 = 30 g/10 m2 ; Pl. (m2 ), plants per m2 ; ns, non significant.

Table 3 Effect of different cultivars (CV) and planting densities (PD) on essential oil (EO) (%), essential oil yield (EOY) (kg/ha), estragol (ES) (%) and trans-anethole (TA) (%) of anise at early and delayed sowing date in Gross-Gerau 2008. CV

PD

Gross-Gerau 2008 1st sowing date (1.04.2008) −2

Pl. (m

1 2 3 PD CV PD × CV

1 2 3 – – –

189 273 315 309 350 117

)

2nd sowing date (17.04.2008)

EOY (kg/ha)

EO (%)

ES (%)

TA (%)

Pl. (m−2 )

EOY (kg/ha)

EO (%)

ES (%)

TA (%)

12.8 10.7 7.9 12.8 12.3 6.4

2.87 2.90 2.30 2.77 2.70 2.60

0.70 0.67 0.67 0.50 0.47 1.07

94.2 94.5 94.4 92.6 93.6 96.9

200 292 324 336 354 126

13.6 13.5 11.5 15.3 15.5 7.7

2.57 2.73 2.70 2.80 2.90 2.30

0.67 0.67 0.63 0.5 0.5 0.97

93.3 93.5 93.6 92.3 92.0 96.2

2.07 2.07 ns

0.12 0.12 0.21

ns 0.05 ns

ns 0.45 ns

ns 2.79 ns

ns 0.24 ns

ns 0.05 ns

ns 0.45 ns

LSD at 5% level: CV 1, Enza Zaden; CV 2, Pharmasaat; CV 3, Hild Samen; PD: 1 = 8 g/10 m2 , 2 = 15 g/10m2 , 3 = 30 g/10 m2 ; Pl. (m2 ), Plants per m2 ; ns, non significant.

H. Ullah, B. Honermeier / Industrial Crops and Products 42 (2013) 489–499

1 2 3

1 2 3 – – –

2nd sowing date (17.04.2008)

Pl. (m−2 )

Table 4 Correlation coefficients for the relationships between the yield and quality parameters of anise in Gross-Gerau 2008. Early sowing date Gross-Gerau 2008 UN 0.028 0.954** –

−0.095 0.262 0.262 –

FY 0.187 0.220 0.157 −0.015 –

Delayed sowing date Gross-Gerau 2008 EO 0.290 −0.237 −0.293 −0.175 0.667** –

ES −0.063 0.574** 0.585** 0.172 −0.345* −0.495** –

TA −0.167 0.201 0.226 0.387* −0.745** −0.691** 0.434** –

DL 0.077 0.229 0.200 −0.065 −0.553** −0.524** 0.495** 0.592** –

UN 0.050 0.996** –

TFW

FY

−0.180 0.162 0.150 –

0.327 0.031 0.020 −0.370* –

EO 0.081 −0.218 −0.219 −0.481** 0.398* –

ES −0.095 0.418* 0.425** 0.505** −0.482** −0.595** –

TA

DL

−0.205 0.005 0.016 0.450** −0.757** −0.500** 0.640** –

−0.233 0.216 0.220 0.263 −0.357* −0.707** 0.661** 0.536** –

PH, plant height; PB, primary branches; UN, umbel number; TFW, 1000-fruit weight; FY, fruit yield; EO, essential oil; ES, estragol; TA, trans-anethole; DL, disease level. * Correlation is significant at p 0.05. ** Correlation is significant at p 0.01.

Table 5 Effect of different cultivars (CV) and planting densities (PD) on plant height (PH) (cm), number of primary branches per plant (PB), number of umbels per plant (UN), number of fruit per plant (FN), 1000-fruit weight (TFW) (g) and fruit yield (FY) (dt/ha at 91% DM) of anise at early and delayed sowing date in Gross-Gerau 2009. CV

PD

Gross-Gerau 2009 1st sowing date (1.04.2009)

1 2 3 PD CV PD × CV

1 2 3 – – –

2nd sowing date (15.04.2009)

Pl. (m−2 )

PH (cm)

PB (No.)

UN (No.)

FN (No.)

TFW (g)

FY (dt/ha)

Pl. (m−2 )

PH (cm)

PB (No.)

UN (No.)

FN (No.)

TFW (g)

FY (dt/ha)

374 542 916 656 526 650

55 54 51 54 54 53

3.6 2.4 1.5 2.5 2.8 2.3

4.8 3.5 2.1 3.6 3.9 3.4

71 36 13 36 50 35

2.54 2.24 1.81 2.14 2.22 2.24

8.2 6.7 5.0 6.3 7.4 6.2

345 402 724 517 387 568

48 49 47 47 49 48

2.5 2.5 1.5 2.1 2.2 2.3

3.7 3.5 2.6 3.1 3.3 3.4

70 63 37 51 68 58

2.30 2.26 2.16 2.15 2.36 2.21

7.1 7.0 5.9 6.1 7.3 6.6

ns ns ns

0.47 ns ns

0.53 ns ns

14 ns ns

0.15 ns ns

1.1 ns ns

ns ns ns

0.67 ns ns

0.73 ns ns

ns ns ns

ns ns ns

ns ns ns

H. Ullah, B. Honermeier / Industrial Crops and Products 42 (2013) 489–499

PH PB UN TFW FY EO ES TA DL

TFW

LSD at 5% level: CV 1, Enza Zaden; CV 2, Pharmasaat; CV 3, Agri-Saaten; PD: 1 = 8 g/10 m2 , 2 = 15 g/10 m2 , 3 = 30 g/10 m2 ; Pl. (m2 ), Plants per m2 ; ns, non significant.

493

TFW

0.127 0.057 0.068 – 0.649** 0.992** – −0.329 −0.721** −0.711** −0.643** −0.621** −0.023 0.495** −0.194 –

DL TA

0.073 −0.265 −0.263 −0.180 −0.203 0.008 –

−0.362* 0.125 0.119 0.146 −0.223 −0.030 −0.632** –

ES EO

−0.134 −0.204 −0.207 0.155 −0.083 –

FY

0.681** 0.554** 0.558** 0.509** – PH PB UN TFW FY EO ES TA DL

PH, Plant height; PB, primary branches; UN, umbel number; TFW, 1000-fruit weight; FY, fruit yield; EO, essential oil; ES, estragol; TA, trans-anethole; DL, disease level. * Correlation is significant at p 0.05. ** Correlation is significant at p 0.01.

TA

−0.193 −0.021 −0.051 0.053 −0.246 0.029 −0.463** – 0.140 −0.137 −0.088 −0.022 0.173 0.173 –

ES EO

0.289 0.688** 0.683** – 0.485** 0.996** –

0.636** 0.268 0.297 0.295 –

UN TFW UN

FY

Delayed sowing date Gross-Gerau 2009 Early sowing date Gross-Gerau 2009

Table 6 Correlation coefficients for the relationships between the yield and quality parameters of anise in Gross-Gerau 2009.

0.084 −0.075 −0.066 −0.184 0.141 –

DL

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−0.045 −0.326 −0.275 −0.146 −0.338* 0.129 0.383* 0.004 –

494

fungal infection was noticed in early sowing date (until: 6.2 level) whereas a lower infection level was observed in delayed sowing date (until: 4.6 level). In 2009 at experimental station Gross-Gerau, higher planting densities were recorded in early and delayed sowing dates (Table 5). Plant height showed non-significant differences with respect to used treatments in both sowing dates; however a decreasing trend of plant height was noticed at higher plant densities (Table 5). The different plant densities significantly affected the fruit yield in early sowing date, but not after delayed sowing. The lower plant densities resulted in the higher number of branches, umbels and fruits per plant and enhanced the fruit yield. The highest fruit yield was obtained from 374 plants m−2 in first sowing whereas no plant density effect was observed in delayed sowing. The number of fruits per plant decreased with increasing planting densities in both sowing dates. It was found that there was a significant effect of planting densities on 1000-fruit weight (TFW) in early sowing date, whereas non-significant differences were observed in delayed sowing date. From both sowing times TFW ranged from 1.81 to 2.54 g (Table 5). The highest TFW of 2.54 g was obtained from the plant densities of 374 plants m−2 , while the lowest TFW of 1.81 g was calculated from the planting densities of 916 plants m−2 (Table 5). Number of branches and number of umbels were affected significantly by different plant densities in both sowing dates (Table 5). Lower planting densities led to higher numbers of branches and umbels per plant. Non-significant differences were observed among used cultivars regarding branches per plant and umbels per plant. In early sowing date fruit number was significantly affected by various planting densities. A positive and significant relationship was found between fruit yield and plant height (r = 0.681 1st sowing date, r = 0.636 2nd sowing date) (Table 6). There were strong significant correlations (r = 0.996 1st sowing date, r = 0.992 2nd sowing date) between the number of primary branches and the number of umbels per plant (Table 6). In 2009, plant height (r = 0.681), number of primary branches (r = 0.554), number of umbels (r = 0.558) were positively associated with fruit yield in early sowing time (Table 6). Contrary to that, in delayed sowing date only plant height (r = 0.636) showed positive and significant correlation with fruit yield. Essential oil yield was significantly influenced by planting densities in early sowing date where higher essential oil yield was recorded for plants obtained from lower planting densities (Table 7). The essential oil contents of anise fruits ranging from 2.69 to 3.30% in both sowing dates were not significantly affected by different planting densities and used cultivars. Independent of that, a higher level of essential oil was accumulated in fruits of delayed sowing date (Table 7). The percentage of trans-anethol in the essential oil of anise ranged from 92.3 to 93.3% in both sowing dates in 2009 (Table 7). In the present study, all essential oil samples contained a low concentration of estragol which was at a very low level of around 0.37 to 0.52% in both sowing dates. Cv. Enza Zaden contained a significant lower concentration of 0.38% (p = 0.001) in 1st sowing date and 0.44% (p = 0.000) in 2nd sowing date of estragol as compared to other cultivars in both sowing dates (Table 7). Contrary to that cv. Agri Saaten synthesized a significant lower level of trans-anethole in early and delayed sowing dates in comparison with other cultivars. In early sowing date there was a CV × PD interaction regarding trans-anethole concentration (Table 7). A significant but negative correlation was noticed between estragol and trans-anethole (r = −0.632) (Table 6). 3.3. Field experiment Giessen 2009 In 2009 anise seedlings emerged after 33 and 28 days in early and delayed sowing date (Table 1). Flowering began 64 and 51 days after emergence for the first and second sowing respectively. Time

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Table 7 Effect of different cultivars (CV) and planting densities (PD) on essential oil (EO) (%), essential oil yield (EOY) (kg/ha), estragol (ES) (%) and trans-anethole (TA) (%) of anise at early and delayed sowing date in Gross-Gerau 2009. CV

PD

Gross-Gerau 2009 1st sowing date (1.04.2009) −2

Pl. (m

1 2 3

1 2 3 – – –

)

374 542 916 656 526 650

PD CV PD × CV

2nd sowing date (15.04.2009)

EOY (kg/ha)

EO (%)

ES (%)

TA (%)

Pl. (m−2 )

EOY (kg/ha)

EO (%)

ES (%)

TA (%)

22.2 18.7 13.6 17.6 19.9 17.0

2.69 2.81 2.73 2.74 2.70 2.79

0.37 0.42 0.45 0.38 0.42 0.43

93.2 92.9 93.0 93.3 93.1 92.7

345 402 724 517 387 568

21.9 21.5 18.2 18.3 23.2 20.0

2.96 3.30 3.05 3.23 3.03 3.05

0.47 0.48 0.52 0.44 0.51 0.51

92.6 92.7 92.8 92.8 92.9 92.3

3.13 ns ns

ns ns ns

ns 0.03 ns

ns 0.26 0.44

ns ns ns

ns ns ns

ns 0.04 ns

ns 0.31 ns

LSD at 5% level: CV 1, Enza Zaden; CV 2, Pharmasaat; CV 3, Agri-Saaten; PD: 1 = 8 g/10 m2 , 2 = 15 g/10 m2 , 3 = 30 g/10 m2 ; Pl. (m2 ), Plants per m2 ; ns, non significant.

Table 8 Effect of different cultivars (CV) and planting densities (PD) on plant height (PH) (cm), number of primary branches per plant (PB), number of umbels per plant (UN), 1000-fruit weight (TFW) (g) and fruit yield (FY) (dt/ha at 91% DM) of anise at early and delayed sowing date in Giessen 2009. CV

PD

Giessen 2009 1st sowing date (1.04.2009)

1 2 3 – – –

1 2 3

2nd sowing date (20.04.2009)

Pl. (m−2 )

PH (cm)

PB (No.)

UN (No.)

TFW (g)

FY (dt/ha)

Pl. (m−2 )

PH (cm)

PB (No.)

UN (No.)

TFW (g)

FY (dt/ha)

39 122 271 156 118 157

70 65 62 65 67 65

– – – – – –

– – – – – –

3.30 3.38 3.25 3.36 3.43 3.14

12.0 9.9 9.0 10.2 10.3 10.5

54 134 258 147 148 151

69 65 62 64 66 65

8.5 7.2 5.6 7.0 7.5 6.9

14.8 10.2 6.2 9.5 11.4 10.5

2.81 2.78 2.62 2.77 2.79 2.66

4.4 4.8 4.2 4.3 4.7 4.4

2.0 ns 3.4

– – –

– – –

ns ns ns

1.47 ns ns

ns ns ns

0.67 ns ns

2.2 ns ns

0.11 0.11 ns

ns ns ns

PD CV PD × CV

LSD at 5% level: CV 1, Enza Zaden; CV 2, Pharmasaat; CV 3, Agri-Saaten; PD: 1 = 8 g/10 m2 , 2 = 15 g/10 m2 , 3 = 30 g/10 m2 , Pl. (m2 ), Plants per m2 ; ns, non significant.

span between the beginning of flowering and full maturation of fruits was 52 and 58 days, respectively. Anise plants were harvested after 156 (1st sowing) and 162 (2nd sowing) days, respectively. There were significant differences regarding fruit yield induced by plant density in early sowing date at experimental station Giessen 2009. The present study showed that the fruit yield increased with lower planting densities of 39 plants m−2 , whereas it led to reduction with higher planting densities of 271 plants m−2 (Table 8). TFW which ranged from 2.62 to 3.43 g was significantly influenced by cultivars and planting densities only in delayed sowing date (Table 8). Number of branches per plant and number of umbels per plant showed significant differences regarding planting densities in delayed sowing date whereas the fruit yield was

not affected significantly by used cultivars and planting densities in delayed sowing. The results of correlation coefficient showed a high correlation (r = 0.855) between the number of primary branches and umbels per plant in delayed sowing date. The results of this study showed low negative correlation between TFW and essential oil content (r = −0.256 and r = −0.135) in early and delayed sowing dates (Table 10). The results of the present study showed that different planting densities induced significant effects on essential oil yield (EOY) in early sowing date. The highest EOY of 41.0 kg ha−1 was obtained from lower planting densities of 39 plants m−2 and the lowest yield of 31.1 kg ha−1 was obtained from narrow planting densities of 271 plants m−2 (Table 9). Essential oil yield increased with lower

Table 9 Effect of different cultivars (CV) and planting densities (PD) on essential oil (EO) (%), essential oil yield (EOY) (kg/ha), estragol (ES) (%) and trans-anethole (TA) (%) of anise at early and delayed sowing date in Giessen 2009. CV

PD

Giessen 2009 1st sowing date (1.04.2009)

1 2 3 PD CV PD × CV

1 2 3 – – –

2nd sowing date (20.04.2009)

Pl. (m−2 )

EOY (kg/ha)

EO (%)

ES (%)

TA (%)

Pl. (m−2 )

EOY (kg/ha)

EO (%)

ES (%)

TA (%)

39 122 271 156 118 157

41.0 32.3 31.1 34.7 34.1 35.6

3.44 3.27 3.46 3.42 3.33 3.42

0.54 0.49 0.46 0.48 0.55 0.46

89.8 90.0 91.4 90.6 90.0 90.6

54 134 258 147 148 151

15.5 16.7 14.0 15.2 15.4 15.4

3.51 3.51 3.36 3.58 3.25 3.55

0.64 0.67 0.70 0.65 0.72 0.65

89.9 89.6 89.7 90.0 89.5 89.7

4.12 ns ns

ns ns ns

0.03 0.03 ns

0.66 ns ns

ns ns ns

ns 0.21 0.37

0.04 0.04 ns

ns 0.34 ns

LSD at 5% level: CV 1, Enza Zaden; CV 2, Pharmasaat; CV 3, Agri-Saaten; PD: 1 = 8 g/10 m2 , 2 = 15 g/10 m2 , 3 = 30 g/10 m2 ; Pl. (m2 ), Plants per m2 ; ns, non significant.

0.060 0.044 −0.032 0.124 0.301 0.151 −0.198 – 0.051 −0.035 −0.056 0.166 –

FY TFW

0.406* 0.233 0.294 – 0.579** 0.855** –

UN

−0.688** – – −0.199 −0.384* −0.144 −0.350* 0.467** – −0.517** – – 0.196 0.014 0.191 −0.649** – 0.491** – – −0.035 0.247 −0.169 – 0.001 – – −0.256 −0.310 – 0.384* – – 0.380* –

DL TA ES EO FY TFW

0.158 – – – – – – PH PB UN TFW FY EO ES TA DL

Delayed sowing date Giessen-2009

UN

In the current study there was a strong variation of fruit yields of anise ranging from minimal 2.4 dt/ha (in early sowing GG 2008) until maximal 12.0 dt/ha (in early sowing at Giessen 2009), which was caused by the treatments as well as by fungal infection (C. malkoffii). Highest variation was observed in Giessen 2009 where fruit yields decreased by three weeks delayed sowing from 9.0 to 12.0 dt/ha to 4.2 to 4.8 dt/ha. This effect can be explained by shortening the juvenile phase (germination to flowering) of anise of 13 days which not only reduced the cumulative temperature but also caused an earlier start to the reproductive phase of the plants. The reduction in the development period due to delayed sowing may have reduced the vegetative growth of plants, which resulted in smaller plants with lower branches and umbels per plant and lead to lower fruit number. Current results confirm the observations of Zehtab-Salmasi et al. (2001) who reported lower anise fruit yield due to late sowing which was related to reduction in vegetative growth of the plants. It can be concluded that delayed sowing resulted in insufficient vegetative growth of anise and plants immediately responded to photoperiod which led to reduced plant length, lower number of umbels per plant and reduced fruit yield (Zolleh et al., 2009; Mirshekari et al., 2011). Contrary to Gi 2009, nearly the same level of fruit yield in both sowing dates was found in GG 2008 as well as in GG 2009. In GG 2008, relatively higher fruit yield was obtained in delayed sowing date compared with early sown anise plants. The small differences in fruit yields between both sowing dates can be explained by the disease infection with C. malkoffii. The lower fruit yield of anise in early sowing date was related to a higher infection rate of C. malkoffii (level 6.2) compared with delayed sowing (infection level 4.6). Overall higher fungus infection was found at experimental station GG which might be explained by higher relative air humidity and higher air temperature during the cultivation period of anise. In the present study, a tendency of higher fungal infection was observed in narrow planting densities compared to lower plant densities. Higher plant density may increase relative humidity within the canopy and increase the duration of leaf wetness by reducing air movement and sunlight penetration. Therefore, it can be assumed that plant density could have significant impact on plant disease incidence in anise (Burdon and Chilvers, 1982; Copes and Scherm, 2005). For both experimental years, an increase in planting density reduced the fruit yield of anise. Current findings are different from the results of Tuncturk and Yildirim (2006) who used different seed rates of 5, 10, 15 and 20 kg/ha and showed a close positive relationship between seed rate and anise seed yield with an optimal

Table 10 Correlation coefficients for the relationships between the yield and quality parameters of anise in Giessen 2009.

4.1. Fruit yield

Early sowing date Giessen-2009

4. Discussion

PH, plant height; PB, primary branches; UN, umbel number; TFW, 1000-fruit weight; FY, fruit yield; EO, essential oil; ES, estragol; TA, trans-anethole; DL, disease level. * Correlation is significant at p 0.05. ** Correlation is significant at p 0.01.

DL TA ES EO

−0.139 −0.288 −0.320 −0.005 −0.001 −0.389* –

planting densities which were related to higher fruit yield. Essential oil concentration of anise fruits ranged from 3.25 to 3.58% for all treatments in both sowing dates. Cultivars showed significant differences regarding essential oil accumulation only in delayed sowing date. Interaction effect CV × PD was observed concerning essential oil percentage in delayed sowing date (Table 9). The major essential oil constituent in the fruits of anise was trans-anethole, comprising 90% of the essential oil. In the early sowing date trans-anethole was affected by different plant densities (Table 9). An increasing trend of trans-anethole was found as plant density increased in 1st sowing date. Opposite results were found in delayed sowing date where plant density had no remarkable variation regarding trans-anethole. A significant but negative correlation was noticed between estragol and trans-anethole (r = −0.649) in early sowing date (Tables 10 and 11).

−0.467** −0.594** −0.534** −0.182 0.095 −0.036 0.232 −0.089 –

H. Ullah, B. Honermeier / Industrial Crops and Products 42 (2013) 489–499

−0.086 0.004 0.137 −0.135 −0.089 –

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497

Table 11 Chemical composition (%) in essential oil of three anise cultivars analyzed by GC–MS at two different experimental stations (means of 4 replications). Compounds

Linalool Estragol Cis anethole Trans-anethole Elemene (delta) Cyclosativene Beta elemene Methyl eugenol ␣-Himachalene ␥-Himachalene ␣-Amorphane (E)-methylisoeugenol ␣-Zingiberene ␤-Himachalene ␣-Muurolene ␤-Bisabolene ␤-Sesquiphellandrene Spathulenol Unknown ␣-Cadinol Unknown Unknown No. Of identified compound. Total a

KIa

1098 1197 1252 1287 1333 1367 1388 1400 1449 1478 1482 1489 1493 1499 1502 1506 1522 1580 1629 1651 1831 1886

Giessen 2008

Gross-Gerau 2008

Enza Zaden

Pharmasaat

Hild Samen

Enza Zaden

Pharmasaat

Hild Samen

0.00 0.32 0.10 79.62 0.54 0.00 0.12 0.00 0.86 8.31 0.17 0.14 0.96 0.53 0.19 0.50 0.10 0.00 0.00 0.10 6.44 1.03 17 100.0

0.00 0.38 0.13 82.09 0.47 0.10 0.12 0.00 0.76 7.39 0.17 0.12 0.85 0.48 0.16 0.43 0.05 0.00 0.00 0.00 5.50 0.80 17 100.0

0.20 0.49 0.06 90.20 0.08 0.00 0.00 0.00 0.13 2.11 0.03 0.12 0.45 0.15 0.03 0.21 0.03 0.00 0.00 0.00 4.95 0.71 16 100.0

0.00 0.35 0.18 84.58 0.36 0.00 0.05 0.00 0.57 5.75 0.13 0.14 0.59 0.36 0.11 0.27 0.00 0.08 0.11 0.07 5.47 0.82 18 100.0

0.00 0.36 0.17 83.80 0.42 0.04 0.11 0.00 0.62 6.18 0.13 0.15 0.71 0.38 0.13 0.31 0.07 0.00 0.13 0.08 5.31 0.92 19 100.0

0.00 0.76 0.07 89.07 0.08 0.00 0.00 0.00 0.15 2.90 0.04 0.07 0.51 0.21 0.00 0.29 0.00 0.00 0.00 0.00 5.23 0.62 13 100.0

KI, Kovat’s retention index.

seed rate of 15 kg/ha. The author further reported that an application of 20 kg/ha seed rate reduced seed yield of anise. But current results agree with the previous work carried out in Germany which showed that narrow planting densities had negative impact on seed yield of anise (Yan et al., 2011). The plant densities of 200 and 374 plants m−2 produced the highest fruit yields in 2008 and 2009, respectively at experimental station Gross-Gerau. Contrary to those results, 39 plants m−2 achieved highest fruit yield at experimental station Giessen. These differences among the studies were probably related to lower plant density which induced higher numbers of yield contributing components which enhance fruit yield. Lowest fruit yields of anise were recorded by cv. Hild Samen at experimental station Gross-Gerau in 2008. This effect could be caused by higher susceptibility of cv. Hild Samen to C. malkoffii compared with other used cultivars. In the present study, fruit yield and plant height were positively and significantly correlated (r = 0.68). Photosynthetic surface in the case of all cultivars resulted in a better source of photosynthates available for grain filling phase (Cosge et al., 2009). 4.2. Morphological and fruit yield components Plant height of anise varied from 40 to 44 cm in GG 2008, from 47 to 55 cm in GG 2009 and from 62 to 70 cm in Gi 2009. This different level of plant length was caused mainly by different soil properties. It can be suggested that higher water holding capacity of the clay soil in Giessen improved plant growth of anise. Additionally, lower germination rate and plant density at this station could be contributed to higher plant length of anise in this experiment. There was a positive correlation between plant height and number of umbels/plant (r = 0.65) which emphasizes the importance of the stem length for formation of morphological parameters and fruit yield. In current trials, only in Giessen 2009 (1st sowing date) plant height was significantly affected by plant densities. The reduction in plant height induced by higher plant densities in both sowing dates was due to higher competition within the plant stand which worsened the growing conditions for anise. Delayed sowing date resulted in smaller plant heights in all experiments. This effect could be due to shorter period of

vegetative growth which is determined by sum of air temperature as well as by photoperiodic conditions (day length). The plant height findings obtained from the study are contradictory to Tuncturk and Yildirim (2006) who reported that anise plant height increased as planting densities narrowed these contradictory results might have been due to climatic variation and genetic traits of the crop plants. The number of branches of anise plant was different in each field experiment, ranging from 2.4 to 4.6 and 1.5 to 3.6 in GG 2008 and 2009 and from 5.6 to 8.5 in Gi 2009, respectively. This effect can be explained by a lower germination rate of anise which was found in Giessen 2009 and by different soil properties, especially the water holding capacity of the soil in both stations. In the current results number of primary branches, umbels, fruits and fruit weight per plant were decreased as plant densities increased. It can be concluded that at a lower plant density, plants have efficient use of available resources such as water, light, and nutrients while at higher planting densities, competition among plants will be more. These findings are in line with the previous work that yield components are decreased by increasing the planting densities (Tunctürk et al., 2005; Tuncturk and Yildirim, 2006; Yan et al., 2011). In executed trials, delayed sowing generally decreased the yield contributing components such as branches, umbels, fruits and fruit weight per plant in both experimental years in GG. These findings are also in conformity with previous work that showed clear decline in number of branches, umbels and fruits per plant due to delayed sowing (Tuncturk and Yildirim, 2006; Yan et al., 2011). Cv. Hild Samen was characterized by higher numbers of yield contributing parameters (branches/plant, umbels/plant, fruits/plant, fruit weight/plant) as compared to other cultivars. It can be suggested that lower plant density of this cultivar reduced the competition for available resources (water, nutrients, light) and increased these yield parameters because more space was available for plant spreading. In executed trials, the number of branches per plant was significantly associated with the number of umbels per plant. Thousand fruit weight (TFW) of anise ranged from 1.81 to 3.43 g among all treatments at both experimental stations. Highest variation was observed in Giessen 2009 which was caused by sowing

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date as well as by plant density and cultivar. Anise plants in delayed sowing plots were characterized by higher fungus infection rate during fruiting stage which contributed to smaller fruit formation. Besides that, a significant effect of plant density on TFW was observed; which was caused by higher competition between anise plants. 4.3. Essential oil According to European Pharmacopeia, anise fruit must have an essential oil concentration higher than 2% (European Pharmacopoeia, 2000). In current field experiments, essential oil content of anise varied from 2.30 to 3.58%. Therefore it can be concluded that anise which is cultivated in Germany (Hessen) meets the demand of the European Pharmacopeia. The level of essential oil in anise fruits was nearly the same as in the field experiments of subtropical regions. Cultivar determined effects on essential oil contents were observed only in GG 2008 (both sowing dates) and in Giessen 2009 (delayed sowing). This effect which caused lower essential oil content of cv. Hild Samen can be due to higher fungus infection of this cultivar which reduced the capacity of oil accumulation. In executed trials, delayed sowing led to significant higher essential oil concentration (p = 0.000) at experimental station GG 2009. Higher essential oil concentration in delayed sowing can be explained by higher temperature (20.2 ◦ C) and lower precipitation (40 mm) occurred during the fruit formation stage (August) of anise, which enhanced essential oil synthesis compared with 2008 where lower temperature (18.4 ◦ C) and higher precipitation (72 mm) occurred during fruiting stage. Zehtab-Salmasi et al. (2001) carried out a study to investigate the effect of water supply and sowing dates on performance and essential oil production of anise in ecological conditions of Iran. The authors showed that essential oil percentage was decreased significantly with delayed sowing, but water limitation resulted in an increase in essential oil contents. Current findings confirm the conclusions that the amount of essential oil produced under drought conditions was either maintained or enhanced, depending on the cultivars and extent of stress (Zehtab-Salmasi et al., 2001; Azizi et al., 2009). Anise essential oil percentage depends not only on genetic resources but also on the development of anise fruits. A significant change in essential oil accumulation was reported during the development of anise fruits with higher values of 5.5% at waxy stage than 3.4% at ripening stage (Omidbaigi et al., 2003; Özel, 2009). In the present study, essential oil contents were lower than waxy stage but similar to that harvested at ripening stage. 4.4. Chemical composition More than 15 different components have been reported in anise essential oil composition (Embong et al., 1977; Lawrence, 1984; Askari et al., 1998; Özcan and Chalchat, 2006; Orav et al., 2008; Yan et al., 2011). In current studies, trans-anethole was the dominant constituent varied from 89.5 to 96.9% of the essential oil which is similar with previous studies of anise (Maheshwari et al., 1989; Askari et al., 1998; Orav et al., 2008; Yan et al., 2011). The minor constituents in anise essential oil from present experiments were similar to that found in previous studies including, ␥-himachalene (2–8%), ␣-himachalene (0.13–0.86%), estragol (0.32–0.76%), cis anethole (0.10–0.18%), elemene delta (0.08–0.54%), ␣-amorphane (0.03–0.17%), ␣-zingiberene (0.45–0.96%), ␤-himachalene (0.15–0.53%), ␣-muurolene (0.00–0.19%) and ␤-bisabolene (0.21–0.50%) (Arslan et al., 2004; Orav et al., 2008; Yan et al., 2011). In current field trials a tendency of higher concentration of trans-anethole was observed in early sowing dates at both experimental stations. Current findings are in agreement with that

of Maheshwari et al. (1989) who reported higher trans-anethole concentrations in early sown anise plants compared with delayedsown plants under ecological conditions of India. This is different from the results of others who showed a marked change of the percentage of trans-anethole essential oil from 90% to 80% as the anise fruit developed from waxy to full mature stage (Omidbaigi et al., 2003). In current trials trans-anethole concentrations were similar in anise fruits harvested at waxy stage in tropical and subtropical regions but higher than fruits harvested at full ripening stage. It can be suggested from current and previous studies that to achieve higher concentrations of trans-anethole, anise fruits should be harvested at ripening stage in temperate conditions whereas waxy stage was recommended to meet higher concentration of transanethole due to higher temperature for tropical and subtropical regions. Estragol, the flavoring agent, is considered to have negative effects on animal and human health and was deleted from the list of flavors in food stuffs (Burt, 2004). In the present study estragol was found in all essential oil samples which varied from 0.37 to 1.07% at both locations. The European Pharmacopeia limit of estragol in essential oil of anise 0.5–6.0% was not exceeded in investigated samples. Cv. Hild Samen was characterized by a higher concentration of estragol and trans-anethole with 0.97–1.07% and 96.2–96.9% respectively at experimental station GG 2008. Orav et al. (2008) analyzed anise fruits from various European countries, samples from Germany named as Germany1 and Germany 2 contained 2.0% estragol and 92.2% trans-anethole and 2.3% estragol and 92.7% trans-anethole respectively. In Giessen 2009, cv. Pharmasaat induced higher concentration of estragol in early and delayed sowing date. These contradictory results were caused by genetic variation of used cultivars. In comparison to previous studies, the values of the present results were not higher than the values reported to be 1.04% (Askari et al., 1998), 0.9–1.5% (Rodrigues et al., 2003), 2.4% (Özcan and Chalchat, 2006), 0.5–2.3% (Orav et al., 2008). A significant but negative correlation was noticed between estragol and trans-anethole (r = −0.632) in early sowing date GG 2009. An increasing trend of trans-anethole was observed as plant density increased in early sowing at experimental station Giessen 2009. This effect may be caused by higher C. malkoffii infection of (4.9 levels) in narrow plant density compared with lower plant density (2.6 levels). Sander and Heitefuss (1998), who conducted experiments with wheat and reported that in response to infection with a compatible race of powdery mildew (Erysiphe graminis f. Sp tritici), levels of phenolic acids slightly increased in leaves of the cv. Syros grown with low and medium nitrogen supply. It can be explained that activity of PAL enzyme which is responsible for the synthesis for phenolic compounds and phenylpropanoids increased under low nutrient level, light (through its effect on phytochrome) and by fungal infection. Fungal invasion triggers the transcription of messenger RNA that codes for PAL that enhances the amount of PAL in the plant, which then stimulates the synthesis of phenolic compounds (Logemann et al., 1995). It can be concluded from current trials and previous studies that secondary metabolites increased when plants faced stressful conditions.

5. Conclusion It can be concluded that anise is sensitive to plant density and sowing date. Under ecological conditions in middle Hessen, in Germany, an early sowing date at the beginning of April with plantings densities of 50–200 plants/m2 concerning experimental sites proves to be a decisive cropping practice in order to increase fruit yield. Delayed sowing causes a yield decrease. Higher sowing rate results in narrow planting densities, but reduces fruit yield

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components, like number of branches, umbels and fruits per plant which leads to lower fruit yield. The cultivars tested in our study showed high essential oil concentrations with more than 90% transanethole and less than 1% estragol and are a good chemotype of anise cultivar. Acknowledgments The authors acknowledge the Higher Education Commission (HEC) Pakistan for providing financial support for PhD studies and Deutscher Akademischer Austauschdienst (DAAD) for arranging the whole stuff in Germany. We are also thankful to the technical staff (Dr. Feng Yan, and Rosa Allerdings) for their considerable assistance. References Adams, R.P., 1995. Identification of Essential Oil Components by Gas Chromatography/Mass Spectrometry. Allured Pub. Crop., IL, pp. 69–351. Arslan, N., Gürbüz, B., Sarihan, E.O., Bayrak, A., Gümüs¸c¸ü, A., 2004. Variation in essential oil content and composition in Turkish anise (Pimpinella anisum L.) populations. Turk. J. Agric. Forest. 28, 173–177. Askari, F., Sefidkon, F., Mirza, M., 1998. Quantitative and qualitative study of components in essential oil of Pimpinella anisum L. J. Pajouhes-Va-Sazandegi 38, 70–77. Awad, N.M., Turky, A.S., Mazhar, A.A., 2005. Effects of bio- and chemical nitrogenous fertilizers on yield of anise (Pimpinella anisum L.) and biological activities of soil irrigated with agricultural drainage water. Egypt. J. Soil Sci. 45, 265–278. Azizi, A., Yan, F., Honermeier, B., 2009. Herbage yield, essential oil content and composition of three oregano (Origanum vulgare L.) populations as affected by soil moisture regimes and nitrogen supply. Ind. Crops Prod. 29, 554–561. Burdon, J.J., Chilvers, G.A., 1982. Host density as a factor in plant disease ecology. Ann. Rev. Phytopathol. 20, 143–166. Burt, S., 2004. Essential oils: their antibacterial properties and potential applications in foods – a review. Int. J. Food Microbiol. 94, 223–253. Copes, W.E., Scherm, H., 2005. Plant spacing effects on microclimate and Rhizoctonia web blight development in container-grown Azalea. Hortic. Sci. 40, 1408–1412. Cosge, B., Ipek, A., Gurbuz, B., 2009. Some phenotypic selection criteria to improve seed yield and essential oil percentage of sweet fennel (Foeniculum vulgare Mill. var. dulce). J. Agric. Sci. 15, 127–133. Dudai, N., Puteivsky, E., Ravid, U., Palevitch, D., Abraham, H.H., 1992. Monoterpene content in Origanum syriacum as affected by environmental conditions and flowering. Physiol. Plantarum 84, 453–459. Embong, M.B., Hadzie, D., Molnar, S., 1977. Essential oil in Alberta anise oil (Pimpinella anisum L.). Can. J. Plant Sci. 59, 681–688. European Pharmacopoeia, 2000. Dritter Nachtrag, 3rd ed. Council of Europe, Strasbourg, pp. 499–500. Farooqi, A.H.A., Sangwan, N., Sangwan, R.S., 1999. Effect of different photoperiodic regimes on growth, flowering and essential oil in Mentha species. Plant Growth Regul. 29, 181–187. Figueiredo, A.C., Barroso, J.G., Pedro, L.G., Scheffer, J.J.C., 2008. Factors affecting secondary metabolite production in plants: volatile components and essential oils. Flavour Frag. J. 23, 213–226. Gülcin, I., Oktay, M., Kirecci, E., Küfrevioglu, Ö.I., 2003. Screening of antioxidant and antimicrobial activities of anise (Pimpinella anisum L.) seed extracts. Food Chem. 83, 371–382.

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Hänsel, R., Sticher, O., Steinegger, E., 1999. Pharmakognosie-phytopharmaize. 6. Auflage. Springer-Verlag, Berlin, Heidelberg, pp. 692–695. Lawrence, D.M., 1984. Progress in essential oils (Anise oil, Calamus oil, Cardamom oils). Perfumer Flavorist 8, 63–65. Logemann, E., Parniske, M., Hahlbrock, K., 1995. Modes of expression and common structural features of the complete phenylalanine ammonia-lyase gene family in parsley. Proc. Natl. Acad. Sci. U.S.A. 92, 5905–5909. Maheshwari, S.K., Gangrade, S.K., Tarivedi, K.C., 1989. Effect of date and method of sowing on grain and oil yield and oil quality of anise. Indian Perfumer 33, 169–173. Mirshekari, B., Hamidi, J., Ramazani, A.Z., 2011. Phenology and yield of cumin at different sowing dates and planting patterns. J. Food Agric. Environ. 9, 385–387. Omidbaigi, R., Hadjiakhoondi, A., Saharkhiz, M., 2003. Changes in content and chemical composition of Pimpinella anisum L. oil at various harvest time. J. Ess. Oil Bearing Plants 6, 46–50. Orav, A., Raal, A., Arak, E., 2008. Essential oil composition of Pimpinella anisum L. fruits from various European countries. Nat. Product Res. 22, 227–232. Özcan, M.M., Chalchat, J.C., 2006. Chemical composition and antifungal effect of anise (Pimpinella anisum L.) fruit oil at ripening stage. Ann. Microbiol. 56, 353–358. Özel, A., 2009. Anise (Pimpinella anisum L.) changes in yields and component composition on harvesting at different stages of plant maturity. Exp. Agric. 45, 117–126. Reineccius, G., 1994. Source Book of Flavours, 2nd ed. Chapman and Hall, New York. Rodrigues, V.M., Rosa, P.T.V., Marques, M.O.M., Petenales, A.S., Meireles, M.A.A., 2003. Supercritical extraction of essential oil from aniseed (Pimpinella anisum L.) using CO2 solubility, kinetics and composition data. J. Agri. Food Chem. 51, 1518– 1523. Sander, J.F., Heitefuss, R., 1998. Susceptibility to Erysiphe graminis f. sp. tritici and phenolic acid content of wheat as influenced by different levels of nitrogen fertilization. J. Phytopathol. 146, 495–507. Tabanca, N., Demirci, B., Kirimer, N., Baser, K.H.C., Bedir, E., Khan, I.A., Wedge, D.E., 2005. Gas chromatographic–mass spectrometric analysis of essential oil from Pimpinella aurea, Pimpinella corymbosa, Pimpinella peregrina and Pimpinella puberula gathered from Eastern and Southern Turkey. J. Chromatogr. A 1097, 192–198. Tabanca, N., Demirci, B., Kirimer, N., Baser, K.H.C., Bedir, E., Khan, I.A., Wedge, D.E., 2006. Gas chromatographic–mass spectrometric analysis of essential oil from Pimpinella species gathered from Central and Northern Turkey. J. Chromatogr. A 1117, 194–205. Tepe, B., Akpulat, A.H., Sokmen, M., Daferera, D., Yumrutas, O., Aydin, E., Polissiou, M., Sokmen, M., 2006. Screening of the antioxidative and antimicrobial properties of the essential oil of Pimpinella anisetum and Pimpinella flabellifolia from Turkey. Food Chem. 97, 719–724. Tirapelli, C.R., Andrade, C.R., Cassano, A.O., De Souza, F.A., Ambrosio, S.R., Costa, F.B., Oliveria, A.M., 2007. Antispasmodic and relaxant effects of the hydroalcoholic extract of Pimpinella anisum (Apiaceae) on rat anococcygeous smooth muscle. J. Enthopharmacol. 110, 23–29. Tunc, I., Sahinkaya, S., 1998. Sensitivity of two greenhouse pests to vapours of essential oils. Entomol. Exp. Appl. 86, 183–187. Tunctürk, M., Ekin, Z., Türközü, D., 2005. Response of black cumin (Nigella sativa L.) to different seed rate growth, yield components and essential oil components. J. Agron. 4, 216–219. Tuncturk, M., Yildirim, B., 2006. Effect of seed rates on yield and yield components of anise (Pimpinella anisum L.). Indian J. Agric. Sci. 76, 679–681. Yan, F., Beyer, E.M., Azizi, A., Honermeier, B., 2011. Effects of sowing time and sowing density on fruit yield, essential oil concentration and composition of anise (Pimpinella anisum L.) under field conditions in Germany. J. Med. Spice Plants 16, 26–33. Zehtab-Salmasi, S., Javanshir, A., Omidbaigi, R., Alyari, H., Ghassemi-golezani, K., 2001. Effects of water supply and sowing date on performance and essential oil production of anise (Pimpinella anisum L.). Acta Agron. Hung. 49, 75–81. Zolleh, H., Bahraminejad, S., Maleki, G., Papzan, A.H., 2009. Response of cumin (Cuminum cyminum L.) to sowing date and planting density. Res. J. Agric. Biol. Sci. 5, 597–602.