Chicken Manure and Phosphorus Influence on Biomass Production and Chemical Composition of the Essential Oil of Ocimum kilimandscharicum

The effects of soil incorporation of five rates of semi-decomposed chicken manure (0, 5, 10, 15 and 20 t ha), with and without the addition of phosphorus (200 kg P2O5 ha) on biomass production and chemical composition of the essential oil from leaves of African blue basil were evaluated. The experimental design was a randomized complete block design in a 5 × 2 factorial scheme, with four replicates. The first cut of plants was performed at 70 days after transplanting (DAT) and the second at 140 DAT. The addition of 20 t ha of chicken manure to the soil induced increase in plants height, fresh and dry mass production and yield of essential oil. The use of chicken manure induced an increase in camphor content and decreased content of 1,8 cineole. After regrowth, biomass production of African blue basil was higher when compared to the first cut.


Introduction
The genus Ocimum (Lamiaceae) consists of approximately 150 species (Bhattacharjya et al., 2019) and is geographically distributed through many tropical and subtropical regions (Caliskan et al., 2017). Ocimum kilimandscharicum Guerke popularly known as "African blue basil" or "Kilimanjaro" is native to East Africa and has been cultivated in many parts of the world. It is a woody shrub, reaching 2.0 m high in temperate regions, propagated either by seeds or cuttings. Seeds are black, small, and once the shrub is established, they can be harvested three times a year, for more than three years (Khare, 2007;Dolly et al., 2012).
In folk medicine, leaves (infusion) of African blue basil has been used for the treatment of constipation, abdominal pain, cough, measles and diarrhoea (Agrawal, 2017) and several pharmacological and biological studies have been previously shown to include antimicrobial (Verma et al., 2011), larvicide and repellent activity (Narwal et al., 2011). This species is rich in essential oil, presenting different chemical composition when collected in different regions or grown on different substrates (Bansal et al., 2018).

Material and Methods
The experiment was carried out at Medicinal Plants Garden of the Federal University of Grande Dourados (UFGD) (-22.195472 o latitude, 54.935694 o longitude and altitude of 430 m), in Dourados-MS and later in the laboratory of medicinal plants (UFGD). The climate of the region is tropical with a dry season of winter (Aw) (Alvares et al., 2014).
Soil chemical attributes as a function of the treatments (after incorporation of chicken manure and phosphorus) were analyzed (Table 1).

Cultivation
Five rates of semi-decomposed chicken manure (0, 5, 10, 15 and 20 t ha -1 ) with and without the addition of phosphorus (200 kg P 2 O 5 ha -1 ), in the form of triple superphosphate, were incorporated into the soil. Treatments were arranged in factorial scheme 5 × 2, in randomized block design, with four replicates. Each plot had a total area of 3.0 m 2 (2.0 m length and 1.5 m width) and harvested area of 2.0 m 2 (2.0 m length and 1.0 m width), with 16 plants arranged in double rows, 0.25 m space between plants and 0.50 m between rows.
Seedling propagation was performed by indirect seeding at 128-cell polystyrene trays filled with Bioplant ® substrate, placed under protected environment by 50% plastic screen. African blue basil seeds were donated by the germplasm bank of Embrapa-Cenargen, Brasília-DF. The plant was identified and exsiccate was deposited at the Herbarium of Universidade Federal da Grande Dourados-DDMS under number 5002.
The area of cultivation was prepared using plough and a leveller harrowing, and then the beds were raised with a rotovator. The triple superphosphate and chicken manure were distributed by hauling and incorporation into the soil, at 0-20 cm depth, one day before transplanting in the appropriate plots.
Seedlings were transplanted when they were about 5 cm high. Cultivation practices in the field consisted of sprinkler irrigation, whenever soil moisture was below 70% of field capacity (which was measured with tensiometer). Control of weeds was performed with hoes between rows and manually within the rows when weeds were about 3 cm high.
During cultivation cycle in the field, the height of all plants of the plots was measured, from 20 to 70 days after transplanting-DAT and from 90 to 140 DAT, at intervals of 10 days. The height of all plants was measured with ruler graduated in centimeters, from ground level to the highest leaf inflection.
At 70 DAT, all plants of the plots were collected by cutting their stems at 10 cm from ground level. After regrowth, a new cut was made at 140 DAT. In each harvest the production of fresh and dry weight of leaves, stems and inflorescences were evaluated by weighing in a digital scale with a precision of 0.1, in addition to the determination of leaf areas using image analyzer Windias 3 (Windias, Delta-TDevices, Cambridge, UK).
For determination of dry weight, the fresh weight of different morphological components were used which, after being separated, was placed in an oven gas forced at 60±2 °C until constant weight and subsequently weighed in a digital scale. Table 1. Chemical attributes of soil samples collected in the experimental area after incorporation of chicken manure and phosphorus (before installing the experiment) and after the second harvest of African blue basil plants (after finishing the experiment) Before After Before After Before After Before After Before After Before After Before After Before After Before After Before After Note. 1 Analyses performed at the laboratory of soils of Faculdade de Ciências Agrárias (FCA)-UFGD.

Extraction and Oil Composition
The essential oil was extracted from 200 g of fresh leaves of plants of each treatment, both in the first and second harvest. Due to the low production of fresh leaves in the first harvest, the repetitions of each treatment were grouped for the extraction of essential oil. The extraction was performed by hydro distillation using Clevenger type apparatus, according to the methodology proposed by Charles and Simon (1990), using 3 L of water, for approximate 4 (four) hours, was dried over anhydrous sodium sulphate and, after filtration, and later reading of the essential oil volume, stored in glass bottles at low temperature (4°C), until the chemical composition analysis. The following formulas were used for calculation: Essential oil content = Volume (mL)/Mass (g) Essential oil yield = Content × Mass (kg ha -1 ) Essential oil analyses were performed by gas chromatography with flame ionization detector (GC-FID) and gas chromatography coupled to the mass spectrometer (GC/MS). Analyses by GC-MS were performed employing a gas chromatograph (GC-17A, Shimadzu, Kyoto, Japão) with mass detector (QP 5050), using a fused silica capillary column DB-5 (J & W, Folsom, California) 5% phenyldimethylpolysiloxane in fused silica capillary (30 m length × 0.25 mm diameter × 0.25 µm thickness). The analyses conditions were: Helium as carrier gas (99.999% and a flow rate of 1.0 mL min -1 ). Injection of 1 µL in split mode (1:20). Oven programing with an initial temperature of 50 °C at 3 °C min -1 until 250 °C. Temperatures of injector, detector and transfer line were 280 °C. The scanning parameters included electron impact ionization voltage of 70 eV, a mass band of 45 to 500 m/z and scan interval of 0.5 s. The retention index of each peak was calculated using a mixture of linear alkanes (C 8 -C 30 ). The identification of compounds was performed by comparison of mass spectra with the equipment library and with the data reported by Adams (2001).
For determination of relative area, gas chromatography with flame ionization detector (ThermoScientific-Focus GC, San Jose, CA, USA) was employed, with capillary column OV-5 (Ohio Valley SpecialtyCompany, Marietta, OH, USA) 5% phenyldimethylpolysiloxane (30 m length × 0.25 mm diameter × 0.25 µm thickness). Injection of 1 µL in split mode (1:20). Oven programming with an initial temperature of 50 °C at 3 °C min -1 until 250 °C. Injector and detector temperatures of 250 °C, using N 2 as carrier gas (99.999% and flow rate of 1.0 mL min -1 ). The chromatograms were registered by Chrom Quest 5.0 program and analyzed by Workstation Chrom Data Review program. jas.ccsenet.

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Results
The most cm), which increase in soil (      factors, nutrition is noteworthy, since deficiency or excess of nutrients can promote greater or lesser production of active principles. When assessing the chemical composition of the essential oil of this same species in India, Verma et al. (2011) also found camphor as a major constituent, with an average content of 63.4%, proving that cultivation site and nourishing of plants influence the content of components of the essential oil. Our results were positive because the predominance of the camphor and limonene was significantly influenced, demonstrates that the studied essential oil could be a good source of these compounds. They were extensively studied, with potential anti-inflammatory and anticancer activity (Banerjee et al., 1995;Chi et al., 2013;D'Alessio et al., 2013;Goel & Roa, 1988;Ghanta et al., 1987;Kim et al., 2013).

Conclusion
This study demonstrated that the addition of 20 t ha -1 of chicken manure to the soil induced increase in biomass and yield of the essential oil of African blue basil. After regrowth, biomass production of African blue basil was greater. In addition, there was a gain in camphor content using chicken manure. However, there was a decrease in the content of 1,8 cineole. Therefore, if the aim is to produce camphor, it is recommended to use of chicken manure, and if the aim is to produce 1,8 cineole, chicken manure should not be used.