Acta Botanica 2-2016 - za web.indd 260 ACTA BOT. CROAT. 75 (2), 2016 Acta Bot. Croat. 75 (2), 260–265, 2016 CODEN: ABCRA 25 DOI: 10.1515/botcro-2016-0025 ISSN 0365-0588 eISSN 1847-8476 Effects of biodynamic production on growth and essential oil content in basil Slavica Dudaš1, Danijela Poljuha2*, Ivana Šola3, Sabina Šegula4, Sanja Varga1, Barbara Sladonja2 1 Polytechnic of Rijeka, Agricultural Department, Poreč, Karla Huguesa 6, Poreč, Croatia 2 Institute of Agriculture and Tourism, Karla Huguesa 8, Poreč, Croatia 3 Department of Biology, Faculty of Science, University of Zagreb, Horvatovac 102a, Zagreb, Croatia 4 Biotechnical Centre Naklo, Strahinj 99, Naklo, Slovenia Abstract – The effects of a biodynamic sowing calendar on the growth (plant height, fresh herb yield, nodes number) and quality (percentage of leaf mass, essential oil content) of three basil species, Ocimum america- num L., Ocimum × hybrida and Ocimum basilicum L., represented by the cultivars ‘Rosso’ and ‘Eco Geno- vese’, were tested. Statistical analyses showed that the species had greater impact on the observed parameters than either the sowing date or the species and sowing date in combination. The species showed a signifi cant infl uence on all fi ve tested parameters, while sowing date and interaction of both factors signifi cantly infl u- enced plant height, leaf percentage and essential oil content. The best yield per plant was obtained for O. × hybrida and O. basilicum ‘Eco Genovese’. The lowest species O. × hybrida produced the highest amount of essential oil. »Nodes number« parameter most clearly separated the species, but not the cultivars. Even though O. americanum gave the tallest plants, it did not yield either the highest amount of fresh herb or es- sential oil. This species’ height was most consistent, considering the signifi cant impact of biodynamic rhythm. Sowing date was not crucial for basil fresh yield; however if there is a need for taller plants with a higher percentage of leaf mass and more essential oil, sowing date needs to be controlled. Key words: biodynamic production, essential oil, Ocimum americanum L., Ocimum basilicum ‘Eco Geno- vese’, Ocimum basilicum ‘Rosso’, Ocimum × hybrida, sowing calendar * Corresponding author, e-mail: danijela@iptpo.hr Introduction The genus Ocimum belongs to the large botanical fami- ly Lamiaceae which includes a high number of aromatic plants, well-known herbs and ornamental plants grown worldwide. Sweet basil (Ocimum basilicum L.) is the most widely grown Ocimum species in the world for the fresh market, but also for essential oil production (Zheljazkov et al. 2008). Sweet basil essential oil is used in medicine, for aromatherapy, and the cosmetic and food industries (Pu- tievsky and Galambosi 1999), and as an insecticide (Ume- rie et al. 1998, Keita et al. 2001, Pascual-Villalobos and Ballesta-Acosta 2003). Due to its insecticide effects, sweet basil is also used in intercropping, for example with cotton (Shader et al. 2005). After more than 200 analyses of essen- tial oils isolated from O. basilicum L., Lawrence (1988) classifi ed essential oils of sweet basil into four main che- motypes: methyl chavicol-rich, linalool-rich, methyleuge- nol-rich, methyl cinnamate-rich, and also found numerous subtypes. O. americanum is not often used as a culinary herb, unlike the related O. basilicum, but more often as a medicinal plant (Vieira et al. 2003). The essential oils in this species have strong fungicidal activity (Dubey 1991), and the leaves have been used as an insecticide against postharvest damage (Weaver et al. 1991). On the global market of agricultural products, organic, safe, high quality agricultural products, including herbs and spices, achieve the best prices. Organic agricultural produc- tion implies, in general, production without easily soluble fertilizers and chemical pesticides. According to EC Reg. 834/2007 it combines best environmental practices, a high level of biodiversity, the preservation of natural resources and the application of high animal welfare standards. The objectives of organic plant production are to work within natural systems and cycles, to maintain soil fertility, use re- newable resources and to produce safe, nutritious, whole- some food (Mikkelsen 2008). The concept of biodynamic agricultural production, de- vised by Rudolf Steiner (1924), respects the principles of organic production, supplemented by the use of biodynamic EFFECTS OF BIODYNAMIC PRODUCTION ON BASIL ACTA BOT. CROAT. 75 (2), 2016 261 preparations (Demeter International Production Standards 2005) and implies the use of a strict biodynamic sowing and planting calendar (Wistinghausen et al. 1998). Namely, it has been proven that time of sowing has a signifi cant ef- fect on plant growth or yield (Ejimofor Ogbonna et al. 2012, Ehsanullah et al. 2014). Biodynamic preparations in- clude strictly prescribed herbal and mineral additives and extracts for plant conditioning and compost activation, bio- stimulants and organic fertilizers, manures and composts (Wistinghausen et al. 1998). The use of biodynamic prepa- rations in organic production is also allowed by EU regula- tion (Council Reg. 834/2007). Organic production in Croatia is a small, but growing, branch of food production; it occupies about 2.5% (Minis- try of Agriculture, 2013) of all farmland and is subsidized by the Ministry of Agriculture. Biodynamic production, however, is not yet recognized as very promising. The aim of this study was to test the effect of four sowing dates rec- ommended in the biodynamic sowing and planting calendar (Thun and Thun 2011) on the growth and essential oil con- tent of three basil species (including two cultivars of one species) grown in pots for fresh seasoning herb. For that purpose we screened: 1) plant height, 2) number of nodes, 3) yield of fresh herb, 4) percentage of leaves mass, and 5) essential oil content of O. basilicum ‘Rosso’, O. basilicum ‘Eco Genovese’, O. × hybrida and O. americanum. Material and methods The practical experiment was conducted in summer 2012 with different basil species/cultivars (X): two culti- vars of sweet basil, O. basilicum ‘Rosso’ and ‘Eco Geno- vese’, bushy basil O. × hybrida, and the citral type basil O. americanum L. The experiment was conducted in a plastic tunnel and on an open area of the Institute of Agriculture and Tourism in Poreč. The organic seeds used in the experi- ment were supplied for O. basilicum ‘Rosso’ and ‘Eco Genovese’ by Amarant (Slovenia) and for O. × hybrida and O. americanum by Reinsaat (Austria). Four species/cultivars were tested and each one was sown on four different sowing dates. Sowing was done un- der the usual climatic conditions for this geographic area and period (monthly average temperatures were for June 23.1 °C, July 26.1 °C, August 25.1 °C; total monthly pre- cipitation was for June 18.6 L m–2, July 2.2 L m–2, August 3.8 L m–2) (Croatian Meteorological and Hydrological Ser- vice, 2012). Basil was sown in containers fi lled with stan- dard substrate, Klasmann 1 (Producer: Klasmann-Deil- mann, Germany) on four different sowing dates (Y) chosen according to the biodynamic rhythm sowing scheme (Thun and Thun 2011), recommended for leafy plants in the Medi- terranean climatic zone: (1) June 7th 2012 after 1.00 pm – suitable for root plants; (2) June 8th 2012 after 6.00 pm – suitable for sowing of fl owering plants; (3) June12th 2012 after 8.00 am – suitable for sowing of leafy plants; (4) June 14th 2012 after 5.00 pm – suitable for sowing of fruity plants. From the scientifi c viewpoint, these sowing periods will be evaluated as different sowing dates with minimal timings. At the 4 true leaf stage, basil plants were transplanted into pots (644 mL volume) in Klasmann 2 (Producer: Klas- mann-Deilmann, Germany) standard substrate. Pot basil was placed in an open area. Irrigation with tap water was done daily, fertilisation twice after transplantation with 2.0 dL per pot of 1.0% standard organic fl uid fertiliser Bio Pl- antella (Producer: Unichem Agro Ltd). Every treatment was represented in three repetitions with 15 pots per repeti- tion. Four species/cultivars (X) were tested and each one was sown in four different sowing dates. Species and cultivars with the same sowing date began to fl ower homogenously, with only 1–2 days difference. The duration of the cultiva- tion was considered as a period in days from sowing to har- vest, where the fi rst day was the sowing day and the last day was the day of harvest. It lasted on average between 44 and 54 days, depending on the sowing date (Tab. 1). The shortest-lasting cultivation period was for the basil sown on June 7th and June 8th, and the longest for the basil sown on June 12th and 14th. The plant height, number of nodes per main stem, fresh yield per plant, leaf mass percentage and the essential oil content were measured. The harvest of basil was done at the beginning of the fl owering period, at the time of the appear- ance of the fi rst fl owers in 10% of plants. Measurements of fresh herb were made just before harvesting. Fresh herb was then air dried in a shady and airy place in standard room conditions. After drying, leaves were separated from the stems; the leaf-to-stem ratio presents the value obtained from the mass of dried leaves divided by the mass of dried stems. Plant height was defi ned as the length of the main stem in cm and the number of nodes was determined by counting from the fi rst fully developed leaves in the basal part of the main stem until the last fully developed and vis- ible nodes. Leaf mass percentage was determined as the percentage of leaves in the whole mass of dried herb. Es- sential oil content was expressed in ml per 100 g of leaf dried weight. The essential oil was distilled with the Neo-Clevenger apparatus, using the method described in the European Pharmacopeia 5.0 (2005). Each sample, represented by three repetitions, was distilled twice. Experimental data were statistically analysed using fac- torial (two way) ANOVA and post hoc Tukey test for mul- tiple comparisons between groups with p ≤ 0.05. Tab. 1. Biodynamic calendar of basil. Sowing date Germination (days after sowing) Transplanting into pots (days after sowing) Flowering beginning (days after sowing) Harvesting date June 7th 4 22 44 July 23rd June 8th 4 23 45 July 23rd June 12th 4 20 52 August 8th June 14th 4 22 54 August 8th DUDAŠ S., POLJUHA D., ŠOLA I., ŠEGULA S., VARGA S., SLADONJA B. 262 ACTA BOT. CROAT. 75 (2), 2016 Results Statistical analysis of tested factors throughout all ob- served parameters showed that the largest source of varia- tion in all parameters was the species, which is the factor with the largest impact (Tab. 2). Plant height, percentage of leaf mass and essential oil content were signifi cantly affect- ed not only by the species/cultivar, but also by the sowing date (Y) and by the interaction between the species/cultivar and the sowing date (X×Y). Shortly before fl owering, basil formed on average be- tween 8.0 and 10.2 nodes per main stem, depending on the species and cultivar (Tab. 3). The highest number of nodes was observed in O. × hybrida, which formed signifi cantly more nodes than other species/cultivars tested. The highest number of nodes and the lowest plant height were found in O. × hybrida, indicating its very short internodes i.e. its compact, bushy habitus. The lowest number of nodes was found in O. basilicum cultivars. The number of nodes was not infl uenced by sowing date, or by interaction of species/ cultivar and sowing date. The fresh yield of basil ranged between 12.5 and 17.2 g/ plant. O. × hybrida had a signifi cantly higher yield than O. americanum or O. basilicum ‘Rosso’ (Tab. 3). The second best cultivar in terms of fresh yield was O. basilicum ‘Eco Genovese’. The yield per plant was not infl uenced by sow- ing date or by interaction of species/cultivar and sowing date. In Fig. 1, the parameters infl uenced by both species/cul- tivars and sowing date, as well as their interaction, are pre- sented. The height of basil plants at the beginning of fl owering was between 18.1 and 38.3 cm (Fig. 1). This large range is a result of the genetic potential of species/cultivars and the different sowing dates, but is also, as the statistical analysis showed, impacted by the interaction between these two fac- tors (Tab. 2). O. americanum dominated in growth. At the last sowing date, it was signifi cantly higher than all the oth- er species and cultivars. Additionally, at all sowing dates, O. americanum was signifi cantly taller than O. × hybrida. Leaf proportion in dried herbs ranged between 45.6 and 73.1% (Fig.1). Signifi cantly the highest leaf mass propor- tion was found in O. basilicum ‘Rosso’ sown on June 7th, and the lowest in ‘Eco Genovese’ sown on June 8th. The most pronounced differences among species/culti- vars were found for essential oil content (Fig. 1), which was predominantly infl uenced by the species (Tab. 2), and to a lower extent by the sowing date, and by the interaction of both factors. The essential oil content of O. × hybrida was signifi cantly higher than that of any of the other species/ cultivars with the same sowing dates, while the lowest es- sential oil content was found in O. basilicum cultivar ‘Ros- so’. The O. × hybrida of the last sowing date reached maxi- mum essential oil content. Discussion Conventional farming is being progressively replaced by more ecology-friendly and sustainable organic agricul- tural production. Basil is grown worldwide for seasoning and for essential oil extraction. For culinary purposes, fresh basil leaves are used; har- vesting takes place at the period with the best leaf/stem pro- portion (over 1.0), mostly reached shortly before or at the beginning of fl owering. For essential oil extraction, basil is harvested in full bloom, because then it has the highest Tab. 2. Impact of analysed factors (species/cultivar (X) and sowing date (Y)) and their interaction (X×Y) on quantitative and qualitative plant parameters (two-way ANOVA). The numbers denote squared total value of the parameter variation caused by species/cultivar, sow- ing date or their interaction. *signifi cant with p ≤ 0.05, DF – degrees of freedom. Source of variation DF Mean square (s2) Plant height (cm) Nodes number Yield (g per plant) Percentage of leaves mass Essential oil content (mL per 100 g DW) Species/cultivar (X) 3 5320.2* 60.4* 245.1* 245.9* 2.3* Sowing date (Y) 3 1704.5* 1.3 22.6 197.3* 0.1* X×Y 9 342.5* 3.2 26.0 120.0* 0.2* Error 222 24.5 2.0 18.4 1.8 0.0 Tab. 3. Infl uence of analysed factors (species/cultivar (X) and sowing date (Y)) and their interaction (X×Y) on quantitative plant parameters. *signifi cant with p ≤ 0.05. Different letters in the same column indicate signifi cant difference. SD – standard devia- tion. Factor Nodes number ± SD Yield of fresh herb (g per plant) ± SD Species/cultivar (X) O. basilicum ‘Rosso’ 8.3±0.6 c 12.5±1.0 c O. basilicum ‘Eco Genovese’ 8.0±0.9 c 15.8±0.9 ab O. × hybrida 10.2±1.0 a 17.2±0.8 a O. americanum 9.5±0.7 b 14.6±0.6 b Sowing date (Y) June 7th 2012 after 3.00 pm 9.1±1.2 a 15.1±1.1 a June 8th 2012 after 8.00 pm 9.1±0.6 a 14.1±1.0 a June 12th 2012 after 9.00 pm 8.9±0.9 a 15.5±2.0 a June 14th 2012 after 5.00 pm 8.9±1.0 a 15.4±0.8 a ANOVA, Tukey test X * * Y X×Y EFFECTS OF BIODYNAMIC PRODUCTION ON BASIL ACTA BOT. CROAT. 75 (2), 2016 263 essen tial oil content (Sifola and Barbieri 2006). Sifola and Barbieri (2006) investigated the cultivation of three basil cultivars, which lasted from the end of May to mid-June. They harvested basil 49 days after transplanting, in the full bloom stage. The full bloom stage, as mentioned above, is considered to be the most appropriate for commercial har- vesting of basil for essential oil production. These two au- thors report the branching of basil plant, a parameter com- parable to the number of nodes per main stem. Higher bud and node numbers can be an indicator of branching forma- tion potential and yield. In their experiment, basil formed between 9.3 and 9.8 branches. This is in conformity with our determined numbers of nodes of between 8.0 and 10.2 per main stem in pot basil (Tab. 3). The range in numbers of nodes in pot basil is affected mostly by the characteristics of the species/cultivar, which is also in accordance with our result. Additionally, we observed that node number signifi - cantly depended on the tested species, but not on the culti- vars. In the study of Frąszczak et al. (2011) on two basil culti- vars grown in small pots in different daily light regimes and different temperatures, a signifi cant infl uence of the culti- var, temperature and photoperiod on the monitored param- eters was found. These results are in accordance with ours, where the species, i.e. genetic factor, was confi rmed as the main signifi cant factor that infl uenced the monitored pa- rameters. In our experiment, the differences between spe- cies/cultivars of basil were much more expressed, thus clar- ifying the intensity of species/cultivar infl uence on the monitored parameters. Strong cultivar infl uence on the yield of basil was also confi rmed by Politycka and Gołcz (2004), who confi rmed that the yield of dry leaves of green basil cultivars was up to 100% higher than that of cultivars with anthocyanin. We further detected that sowing date itself, as well as the interaction of species/cultivar and sowing date, signifi - cantly affected the plant height, leaf share and the essential oil content of basil, but not the number of nodes and overall yield (Tab. 2). The quality of herbs is, among other factors, infl uenced by the proportion of leaves and stems (Werker et al. 1993, Ioannidis et al. 2002), as well as by essential oil content (Rey and Saez 2002). A higher proportion of leaves than stems represents a higher quality, as the concentration of oil glands is the highest in mature leaves (Werker et al. 1993, Ioannidis et al. 2002) and in basil, leaves and calyxes have the highest concentration of oil glands (Putievsky and Galambosi 1999). The leaf proportion in our experiment is analogous to the leaf-to-stem ratio used by Sifola and Barb- ieri (2006). The leaf-to-stem ratio in their investigation var- ied between 0.7 and 1.2. All values over 1.0 mean that the amount of leaves is over 50%. The percentage of leaf mass in our investigation depended on the species/cultivars, but it was also infl uenced by the sowing date and the interaction of species/cultivar × sowing date (Tab. 2). Only in O. basi- licum ‘Eco Genovese’, which was sown on June 8th (second sowing date), was the percentage of leaves under 50%, while all other combinations of species/cultivars and sow- ing dates had leaf percentages of over 50%. Essential oil content, as the second quality aspect, in four basil species and cultivars, ranged between 0.4 and 1.8 mL per 100 g dried leaves, depending on the species/culti- var and sowing date (Fig. 1). The species with the highest amount of essential oil in this study was O. × hybrida, while O. basilicum ‘Rosso’, as expected, had the lowest amount. Interestingly, even though O. × hybrida had the least plant height, it contained the highest amount of essential oil, indi- cating that internodes negligibly contribute to essential oil production. O. basilicum ‘Rosso’ is a cultivar for decorative purposes; it is purple basil with high anthocyanidin content. Compared to the essential oil content in O. basilicum culti- vars from the experimental garden in Prague (Klimánková et al. 2008), our O. basilicum ‘Rosso’ is most similar to the Czech cultivar V, while O. basilicum ‘Eco Genovese’ is more similar to the Czech cultivars I–IV. Based on the obtained data we conclude the following: 1) species had greater impact on the observed parameters than sowing date or species and sowing date in combina- Fig. 1. Plant parameters infl uenced by both species/cultivar and sow- ing date, as well as their interaction: A) height, B) leaf mass and C) essential oil. Data are the means of three replicates ± SD (stan- dard deviation). The means labelled by different letters are signifi - cantly different (two way ANOVA and Tukey’s test), p ≤ 0.05. DUDAŠ S., POLJUHA D., ŠOLA I., ŠEGULA S., VARGA S., SLADONJA B. 264 ACTA BOT. CROAT. 75 (2), 2016 tion; 2) three parameters – plant height, percentage of leaf mass and essential oil content – were also signifi cantly af- fected by the sowing date and the interaction between spe- cies/cultivars and sowing date; 3) the best yield per plant was given by O. × hybrida and O. basilicum ‘Eco Gen- ovese’; 4) the highest production of essential oil was in the lowest species O. × hybrida indicating that Ocimum stem negligibly contributes to the essential oil production. The lowest essential oil content was confi rmed in the O. basili- cum purple cultivar ‘Rosso’; 5) the percentage of leaf mass was over 50.0% in all cases, except in O. basilicum ‘Eco Genovese’, sown on June 8th, with 45.6%; 6) the most clear species separation was according to the »number of nodes« parameter, while the cultivars did not differ; 7) even though O. americanum gave the tallest plants, it did not yield the highest amount of fresh herb or essential oil; 8) the species with the most consistent height, considering the signifi cant impact of biodynamic rhythm, was O. americanum; 9) fi - nally, if we were interested in yield only, then the sowing date would not be crucial, however if we needed higher plants with a higher percentage of leaf mass and more es- sential oil (i.e. more leaves) then the sowing date should be controlled. 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