1. bookTom 30 (2021): Zeszyt 1 (March 2021)
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Otwarty dostęp

Impacts of Environmental Variations on Quality and Chemical Contents of Oriental Tobacco

Data publikacji: 20 Apr 2021
Tom & Zeszyt: Tom 30 (2021) - Zeszyt 1 (March 2021)
Zakres stron: 50 - 62
Otrzymano: 13 Feb 2020
Przyjęty: 08 Mar 2021
Informacje o czasopiśmie
License
Format
Czasopismo
eISSN
2719-9509
Pierwsze wydanie
01 Jan 1992
Częstotliwość wydawania
4 razy w roku
Języki
Angielski
INTRODUCTION

In addition to their characteristic properties, Oriental tobaccos can show a wide variety of characteristics resulting from differences in environmental conditions and cultivation techniques. The quality concept for these tobaccos includes technological characteristics as well as aspects of consumption demand. Therefore, tobacco quality, which is a subjective phenomenon, varies depending on the type of product to be manufactured and the preference of the consumer. The quality of a leaf represents the characteristic of a tobacco type.

The leaf grows depending on the processes undertaken, from planting with the genotype × environment interaction, until the end product in the factory. The quality of a green leaf reaches its potential with subsequent curing, ageing and the manufacturing processes. Therefore, each tobacco type is considered a region-specific ecotype.

Temperature and precipitation are the main environmental variables affecting the performance of plant growth, productivity and chemical composition of tobacco plants (1). Nicotine and soluble carbohydrates contents have been reported to be positively related to the temperature, and negatively to the precipitation (2). These studies indicate that the quality has a positive relationship with drought resistance and flowering earliness, while having a negative relationship with leaf yield and disease resistance (3). Therefore, the aroma and basic quality characteristics of Oriental tobaccos depend largely on the soil characteristics and climate of the region where they are grown (4).

Long-time cultivation of autogamous plant populations in the same region is very important for the selection in breeding. It is significant in the cultivation of pure lines, most variations of which occur due to the effects of environmental conditions. Landraces consisting of different genotypes due to human selection, are a valuable selection material for breeders because of the improvement in their adaptability (5). Genetic variability in a population is important also for biodiversity. Adaptability of a tobacco population to changes in environmental conditions without variability is difficult and it thus becomes more prone to genetic fixation (6).

An increase in the starch content of the leaf also enhances the leaf thickness (7). Increasing the number of upper leaves over time, to protect the lower leaves in Oriental tobacco, is explained by this way of adaptation mechanism. The ability of Oriental tobacco of maintaining the physical and biological properties as well as yield potential in arid ecosystems can be attributed to the high efficiency in use of water and nitrogen of these tobacco types. Environmental influences also stimulate the plants to conserve their performances by developing biochemical and morphological mechanisms, which create quality attributes that distinguish Oriental tobaccos from others. In other words, the quality criteria of Oriental tobaccos, such as leaf size, color, tip angle, hygroscopicity and thickness, are the characteristics generated during the adaptation process against stressors (8).

Alkaloids are known for their direct impact on tobacco quality and usability. Nicotine, the most abundant alkaloid in tobacco among more than 20 other alkaloids, is the essential component that leads to consumption of tobacco products worldwide (9). Other tobacco alkaloids with less pharmacologically active substance content and impact are nornicotine, anabasine and anatabine (10). Glucose and fructose are the most important soluble sugars and are called reducing sugars (11, 12). The leaves with higher reducing sugar content are preferred for tobacco products (13). Sugars are known to balance the smoke flavor primarily by abating the sensory effects of nicotine and other tobacco alkaloids (14). A typical American blend contains 3 to 15% Oriental tobacco, and the total sugar content of the blend is approximately 12% which is composed of 8% natural and 4% additional sweeteners (11).

Polyphenols that determine the color, flavor and smell of tobacco (16) are the most important secondary metabolites due to their containing tannins, coumarin, flavonoids and their derivatives (15). Polyphenol content is important in tobacco processing, as well as in blending and quality control processes (9). Polyphenols are vital components (18, 19) due to their contribution to the sensory properties as well as to color, flavor, and smell of tobacco leaf and in addition to their antioxidant properties (17). Polyphenolic ingredients are sensitive to genotype and environment. Therefore, determining the polyphenol contents of a blend and grades of tobacco is very important (20). The impact of environmental and growing conditions on the polyphenol content of tobacco is higher than the genotype effect. The main polyphenols in tobacco are chlorogenic acid and rutin, their combustion products called catechols are known to be carcinogenic (16, 21, 22). Therefore, the reliable characterization of chemical structures in the definition of leaf tobaccos is necessary for blenders in order to determine the potential toxicity risk (23, 24).

MATERIAL AND METHODS
Material

In 2015 Basma tobacco types in different growing regions of Turkey were screened, morphologically different plants were identified and seeds were collected. The DNA fingerprint analysis of the collected seeds indicated 27 different tobacco lines (Figure 1). Plant material of the study consisted of 21 Basma tobacco lines from the 27 identified lines and four standard tobacco lines (Xanthi 2A, Xanthi 81, Nail and Canik 190–5) (25). Selected plants were isolated using a paper bag to prevent outcrossing. Self-pollinated seeds were harvested after they reached maturity.

Figure 1

Dendrogram showing the genetic affinity of tobacco genotypes.

Setting up the experiment

Seedlings of the 25 genotypes were grown in a float tray system with a peat medium. Composite fertilizer containing 20% nitrogen (N), 10% phosphor (P) and 20% potassium (K) plus micronutrients (iron 0.4%, manganese 0.4% and zinc 0.4%) was mixed with 500 g/m3 water in float pond water to supply nutrients for the seedlings. The experimental fields, where the study material could be cultivated, were chosen in different altitudes. The research was carried out in Evciler (40°36′43.48″N, 36°36′5.25″E, 581 m altitude) and Karayaka (40°44′16.45″N, 36°33′58.31″E, 302 m altitude), two villages of the Tokat-Erbaa, as well as in Bafra, a town in Samsun Province (41°33′45.29″N, 35°52′18.35″E, 26 m altitude) and in Gümüşhacıköy, a town in Amasya Province (40°53′1.03 ″N, 35°12′47.98E, 848 m altitude). Prior to planting the seedlings, 60 kg/ha N, 40 kg/ha P2O5 and 60 kg/haK2O were applied to the experimental fields (26). The field experiments were carried out in a randomized block design with three replications. The seedlings were planted with 45 cm inter-row and 12 cm intra-row spacing on 5-m long plots.

The seedlings were planted on May 21, 2017 in Evciler, May 19, 2017 in Karayaka, July 4, 2017 in Bafra and June 29, 2017 in Gümüşhacıköy. The harvest of mature leaves was completed in three different intervals, each of which was completed in one day.

The leaves were sun-cured, they were threaded onto needles and string and were hung under direct sunlight after being kept in shade for two days. The cured tobacco was baled and stored in a closed room. Data obtained in the different locations were not homogeneous; therefore, the locations were separately subjected to variance analysis using the SAS 9.0 software (59). Duncan's multiple comparison test was used to compare the parameters obtained in different locations and they were graphed with GraphPad Prism 8 program. Soils in the Evciler experimental field had a clay-loam texture and the other three locations had a sandy loam texture. Electrical conductivity levels indicated a very low soil salinity. All soils were slightly alkaline and the highest organic matter content was recorded in the experimental field of Gümüşhacıköy. Soils in Bafra and Karayaka fields had low and Evciler had very low organic matter content. Soils in the Evciler, Gümüşhacıköy and Bafra locations were moderately calcareous while soil in the Karayaka location was calcareous. Potassium contents of all experimental fields were high. Phosphorus contents of the experimental field in Karakaya were moderate while other three fields were low in phosphorus content (Table 1).

Soil analysis results of the locations.

Properties Locations

Evciler Karayaka Gümüşhacıköy Bafra
P2O5 (kg ha−1) 0.51 low 0.62 moderate 0.48 low 0.34 low
K2O (kg ha−1) 16.97 high 17.53 high 15.68 high 13.72 high
Lime (%) 10.2 moderately calcareous 2.39 calcareous 5.17 moderately calcareous 12.73 moderately calcareous
Org. Mat. (%) 95 very low 143 low 2.36 moderate 1.76 low
pH 7.99 slightly alkaline 7.81 slightly alkaline 7.98 slightly alkaline 7.61 slightly alkaline
EC (dS m−1) 0.25 very low 0.13 very low 1.12 very low 0.72 very low
Texture clay loam sandy loam sandy loam sandy loam

The temperature values during the seven-month period covering seedling, field and curing periods of the tobacco were similar to long-term averages, while the relative humidity values were higher than the long-term average values. The average relative humidity during this period was 69.86% in Erbaa, 64.43% in Gümüşhacıköy and 82.24% in Bafra. Total precipitation during the period of vegetative growth was 222.2 mm in Erbaa, 256.5 mm in Gümüşhacıköy and 222.1 mm in Bafra. Compared to the long-term average, there was less rain by 36.1 mm in Erbaa, by 47.1 mm in Gümüşhacıköy and by 152.2 mm in Bafra (Table 2).

Monthly climate data during vegetative growth in the locations.

Months Erbaa Gümüşhacıköy Bafra



LTA. 2017 D. LTA. 2017 D. LTA. 2017 D.
April °C 14.2 12.4 −1.8 11.0 10.0 −1.0 11.2 9.9 −1.3
mm 55.4 45.2 −10.2 56.9 43.6 −13.3 57.7 63.0 5.3
% 58.6 65.2 6.6 58.7 62.9 4.2 79.0 85.8 6.8
May °C 18.1 17.0 −1.1 14.4 14.4 0 15.5 15.0 −0.5
mm 62.2 50.5 −11.7 75.5 70.9 −4.6 47.2 53.6 6.4
% 60.7 71.6 10.9 59.6 69.5 9.9 79.4 87.6 8.2
June °C 21.6 21.4 −0.2 18.2 18.4 0.2 20.1 20.3 0.2
mm 48.4 94.5 46.1 74.9 87.3 12.4 34.6 45.9 11.3
% 58.1 73.6 15.5 58.9 79.2 20.3 74.9 84.7 9.8
July °C 23.9 20.8 −3.1 20.7 21.3 0.6 22.8 23.5 0.7
mm 24.6 1.2 −23.4 26.6 8.4 −18.2 31.0 0 −31
% 55.4 74.2 18.8 53.3 55.5 2.2 73.1 95.2 22.1
August °C 23.8 25.9 2.1 21.1 22.3 1.2 22.9 24.3 1.4
mm 9.9 1.0 −8.9 11.3 9.2 −2.1 47.3 24.8 −22.5
% 55.6 65.8 10.2 50.9 67.7 16.8 74.5 75.2 0.7
September °C 20.5 22.2 1.7 17.4 20.8 3.4 19.5 21.2 1.7
mm 16.1 2.9 −13.2 13.0 13.2 0.2 59.7 13.0 −46.7
% 57.9 61.6 3.7 53.9 51.7 −2.2 76.8 75.2 −1.6
October °C 15.4 13.9 −1.5 12.3 12.2 −0.1 15.4 15.4 0
mm 41.7 26.9 −14.8 45.4 23.9 −21.5 96.8 21.8 −75
% 63.2 77.0 13.8 61.4 64.5 3.1 78.7 72.0 −6.7
Mean temperature (°C) 19.6 19.1 −0.6 16.4 17.1 0.6 18.2 18.5 0.3
Total precipitation (mm) 258.3 222.2 −36.1 303.6 256.5 −47.1 374.3 222.1 −152.2
Mean relative humidity (%) 58.5 69.9 11.4 56.7 64.4 7.8 76.6 82.2 5.6

LTA.: long term average (1963–2016); D: difference between 2017 and the years of 1963–2016;

°C: temperature; mm: precipitation; %: relative humidity

INVESTIGATED CHARACTERISTICS
Grade index

The grade index is the quality score of organoleptic assessments carried out by a tobacco expert on the cured leaves rating properties as stalk position, leaf size, texture, odor, moisture condition, leaf shape, leaf tip angle, leaf venation, color and brightness status, leaf integrity, degradation and disease/pest status of leaves. The expert provides two-digit ratings for each tobacco unit, the first digit representing the stalk position and the second digit representing the quality group according to the aforementioned properties. The grade, which is the basis for the pricing of the product, is calculated by multiplying these degrees with a certain coefficient. The principle of grading is to determine the proportion of A grade tobacco in a certain amount of tobacco (American grading method). The grading is the key action that governs the direction of the product in all stages from processing, marketing and finally to the end product (27).

Nicotine content

For nicotine analysis (% DM), a 200-mg ground moisture-free tobacco sample was weighed into a 50-mL Falcon tube and 1% aqueous acetic acid and acetonitrile (85:15, v/v) was added. The mixture was kept in an ultrasonic water bath for 30 min. Samples removed from the water bath were centrifuged at 4000 rpm for 10 min. The remaining solution (supernatant) atop the precipitated sample was taken up by a syringe. The supernatant was passed through the filter (Nylon, 0.45 μm) and transferred into the vial marked with the sample number. Extractions were analysed using an Agilent technology 1260 series high-performance liquid chromatography (HPLC) system (Agilent Technologies, Boeblingen, Germany) equipped with an ACE C18 column (250 × 4.6 mm i.d. dimensions and 5 μm particle size) with a flow rate of 1 mL/min, a column temperature of 35 °C and a DAD (diode array detector) detector. The mobile phase consisted of 1% acetic acid in water (solvent A) and acetonitrile (solvent B). A wavelength of 324 nm was set for UV detection of alkaloids and nicotine was quantified using an authentic standard (28, 29).

Reducing sugar (glucose and fructose) content

For glucose and fructose analysis (% DM), 1 g of ground moisture-free tobacco sample was weighed into a Falcon tube and 1% acetic acid and methanol (75:25, v/v) was added to the weighed sample. After adding the solvents, the mixture was stirred, then placed in an ultrasonic water bath and kept there for 30 min. The samples were removed from the water bath and centrifuged at 4000 rpm for 8 min.

The remaining solution (supernatant) atop the precipitated sample was subsequently removed with a syringe. The supernatant was passed through a filter (Nylon, 0.45 μm) and transferred into the vial marked with the sample number. The content of glucose and fructose was analysed by HPLC using a RID (refractive index detector) with a Zorbax Carbohydrate column (250 × 4.6 mm i.d. dimensions and 5 μm particle size) at 1.5 mL flow and 40 °C column temperature (29, 30). The mobile phase consisted of 1% acetic acid in water (solvent A) and acetonitrile (solvent B) with 25% A and 75% B.

Phenolic (chlorogenic acid and rutin) content

For chlorogenic acid and rutin analyses, 200 mg ground moisture-free tobacco sample was weighed into a Falcon tube and 5% acetic acid and methanol (85:15, v/v) was added into the tubes. After adding the solvents, the mixture was stirred, placed in an ultrasonic water bath and kept there for 30 min. Samples were removed from the water bath and centrifuged at 4000 rpm for 10 min. The remaining solution (supernatant) atop the precipitated sample was removed with a syringe. The supernatant was passed through the filter (Nylon, 0.45 μm) and transferred into a vial marked with the sample number. The extract was analysed by HPLC with a poroshell 120 EC C18 column (2.7 μm, 150 mm × 3.0 mm i.d.) and a guard precolumn using a diode array detector (DAD) at a 0.3 mL/min flow rate and a 35 °C column temperature. The mobile phase consisted of 1% acetic acid in water (solvent A) and acetonitrile (solvent B) using an isocritical elution with 85% A and 15% B (20, 29).

The peaks obtained from the sample chromatograms were identified by comparison with the peaks obtained from the standards and the area of each peak was calculated according to their standard calibrations (r2; 0.999 and 1.0). The concentrations of nicotine, glucose and fructose were given as percent (% DM) and those of chlorogenic acid and rutin as ppm. Extraction recovery rates, that indicate the reliability of the analysis, were 101% for nicotine, 106% for glucose, 102% for fructose, 83% for chlorogenic acid and 96% for rutin.

RESULTS AND DISCUSSION
Quality grade index

Pricing of tobacco marketed for its degustative properties is practically based on organoleptic estimates, which is a subjective assessment (31). The products with a 60% or higher grade A evaluation obtained the highest market price ($ 4.86/kg) during the study season in 2017. Therefore, genotypes providing a 60% grade A rating have been categorized as high-quality. The A-grade rates of ERB-9, ERB-11, ERB-18, ERB-25 and ERB-27 lines were lower than 60%. Despite the slight differences in the grade values of the remaining 20 genotypes, they were all very alike in quality and commercially on the same scale (Table 3, Figure 2).

Quality grade index and nicotine contents of 25 genotypes in four locations.

Genotypes Quality grade index (%) Nicotine (% DM)


Evciler Karayaka G.Hacıköy Bafra Mean Evciler Karayaka G.Hacıköy Bafra Mean
ERB-5 96.09 ad 74.96 ae 65.05 fi 69.27 a 76.34 0.71 fg 1.81 dh 1.02 bd 0.34 fg 0.97
ERB-6 90.97 ce 75.77 ad 71.75 dg 50.87 ae 72.34 1.02 af 1.28 gh 1.03 bd 0.32 fg 0.91
ERB-7 96.77 ac 79.93 ab 73.07 df 62.36 ac 78.03 1.16 ad 2.02 bg 0.59 hi 0.52 be 1.07
ERB-9 80.06 gh 64.64 bg 43.63 k 39.37 df 56.92 0.87 cg 2.04 bg 0.78 di 0.31 g 1.00
ERB-11 73.20 i 48.92 gh 69.00 eh 39.18 df 57.57 0.88 bg 1.49 fh 0.91 cg 0.46 bg 0.93
ERB-12 96.87 ac 68.19 af 65.13 fi 56.00 ad 71.55 0.82 dg 2.69 ac 0.63 gi 0.61 ac 1.19
ERB-13 98.55 ab 69.60 af 79.93 ad 66.52 ab 78.65 1.32 a 2.59 ad 0.86 ch 0.68 a 1.36
ERB-14 91.77 ce 61.81 cg 58.53 hj 57.65 ad 67.44 1.17 ac 2.58 ad 0.68 fi 0.48 bg 1.23
ERB-15 100.00 a 65.66 bf 88.26 ab 57.10 ad 77.75 0.96 bf 2.30 be 0.92 cg 0.57 ad 1.19
ERB-16 89.22 ef 61.55 cg 76.78 ce 40.47 df 67.01 0.77 eg 1.09 h 1.14 ac 0.41 dg 0.85
ERB-17 85.31 fg 61.30 cg 47.71 k 46.47 be 60.20 1.16 ad 1.69 eh 0.96 cf 0.41 dg 1.06
ERB-18 76.55 hi 61.04 dg 53.04 jk 32.97 ef 55.90 0.83 cg 2.01 cg 1.13 ac 0.47 bg 1.11
ERB-19 90.49 df 83.53 a 87.37 ac 57.41 ad 79.70 0.83 cg 2.07 bf 0.86 ch 0.49 bf 1.06
ERB-21 80.50 gh 65.54 bf 85.38 ac 59.05 ad 72.62 1.05 ae 1.57 eh 0.93 cg 0.36 eg 0.98
ERB-23 95.12 ad 65.72 bf 64.96 fi 67.54 a 73.34 1.14 ad 2.70 ac 0.94 cf 0.40 dg 1.29
ERB-25 76.35 hi 57.89 fg 60.80 hj 44.09 ce 59.78 0.99 fg 1.33 fh 0.92 cg 0.46 bg 0.92
ERB-26 94.35 ae 78.73 ab 57.80 ij 48.99 ae 69.96 0.95 bf 2.52 ad 0.74 di 0.47 bg 1.17
ERB-27 76.07 hi 40.33 h 46.33 k 24.17 f 46.72 0.85 cg 1.53 eh 0.73 di 0.46 bg 0.89
ERB-30 93.33 be 70.14 af 89.89 a 48.87 ae 75.56 1.06 ae 1.51 fh 1.35 a 0.45 bg 1.09
ERB-35 81.44 gh 79.14 ab 85.21 ac 55.77 ad 75.39 0.70 fg 1.99 cg 0.53 i 0.60 ac 0.96
ERB-38 96.65 ac 77.77 ac 84.23 ac 53.72 ad 78.09 1.03 af 2.66 ac 0.86 ch 0.44 cg 1.25
Xanthi 2A 96.84 ac 82.19 a 78.73 be 50.53 ae 77.07 0.99 af 2.81 ab 0.99 be 0.57 ad 1.34
Nail 94.62 ae 68.16 af 62.19 gj 61.33 ac 71.58 1.08 ae 2.58 ad 0.78 di 0.40 dg 1.21
Canik 190–5 94.75 ae 58.94 eg 68.70 eh 59.43 ad 70.45 0.54 g 1.40 fh 1.25 ab 0.42 dg 0.90
Xanthi 81 95.81 ad 79.72 ab 76.68 ce 58.46 ad 77.67 1.22 ab 3.15 a 0.70 ei 0.62 ab 1.42
Mean 89.67 68.05 69.61 52.30 69.91 0.96 2.06 0.89 0.47 1.09
M. checks 95.50 72.26 71.57 57.44 74.19 0.96 2.48 0.93 0.50 1.22
M. lines 88.56 67.25 69.23 51.32 69.09 0.97 1.97 0.88 0.46 1.07
LSD0.05 4.97 13.77 9.30 17.06 0.28 0.66 0.25 0.14
CV (%) 3.37 12.32 8.13 19.87 17.97 19.65 17.14 18.80
Mean square and significance df
Genotype 207.26** 336.07** 564.63** 364.88** 0.10** 0.98** 0.12** 0.03** 24
Error 9.15 70.31 32.09 107.99 0.03 0.16 0.02 0.01 48

Values followed by different letters in each column are significantly different (* p < 0.05) according to Duncan's test; LSD: least significant difference; CV: coefficent of variation; df: degree of freedom;

p < 0.01; Quality grade index: % of A grades; M. checks: mean of checks; M. lines: mean of lines.

Figure 2

Variation in quality grade index (% of A grades) values of 25 tobacco genotypes at four different locations.

The yield and grade values had an inverse relationship, indicating that factors such as soil and climate contributing to the yield increase, had a negative impact on quality (3, 32). The highest grade values and the lowest yield values were obtained in the Erbaa-Evciler location which corroborates the aforementioned inverse relationship between yield and quality grade. Environmental effects induce the plants to maintain their performance by developing biochemical and morphological mechanisms, which are responsible for the quality characteristics that distinguish Oriental tobacco from others (4, 8).

Rainwater contributes to the growth of more elastic and aromatic leaves in Gümüşhacıköy. This location benefits from more regular and continuous rainfall compared to the other locations and it is located in the highest altitude among all the locations (33).

The highest-quality tobaccos were grown in class II and III agricultural land, while the quality was lower in I class land (34, 35). Karayaka was the location where the best quality grade values were obtained due to the effects of the aforementioned factors. The Bafra location has class I agricultural land, therefore this location was included in the study. Water requirement of Oriental tobaccos for a maximum yield is around 400 to 600 mm depending on the length and the climate of the growing season. About 50% of the total water required in the field should be applied during the period of 30–40 days after planting to obtain healthy and resistant plants. The water demand rises to the highest level at 50–70 days after transplanting, and afterwards gradually decreases (36). Excessive irrigation increases the total leaf area, causes thinning of leaves, reduces leaf density and results in quality losses (33).

Color in tobacco is used as a guide in all stages, and defined as the maturity index in cases such as determining the harvest time of stalk positions. The most apparent characteristic of the change in parallel to the advancement in maturity is transformation of leaf colors to yellow, orange and brown following the breakdown of chlorophyll. The vitality, brightness and lightness or darkness of leaf color are important criteria that determine the quality of a particular tobacco type (37). The plants in turgor, due to the continuous water supply, grow more rapidly, and thus the leaves cannot exhibit the type characteristics. In this case, the product will be fine-textured, very low or flavor-free, easily burning, of low-nicotine content and light-colored, which is markedly different from the color of plants grown under regular circumstances (33). The leaf color of Samsun type tobaccos changes from yellow to dark red as cultivation locations are progressing from the lowland to the slopy lands. The curing period of tobacco plants grown in lowland is usually shorter and the color of leaves quickly turns into yellow due to the faster curing (38). Therefore, mostly B grade and partially Kapa group products have been produced in Bafra location.

Nicotine content of leaves

Each type of tobacco within a blend has defining effects on the physical and chemical quality composition, as well as reducing/increasing effects on the amount of nicotine. This fact also has a significant impact on the market demand for the Oriental tobacco types. The desired nicotine content in Basma type tobaccos in the market for the Turkish private sector is in the range of 2.00 to 2.75% DM (26). The nicotine content of tobacco grown in the Karayaka location meets this requirement (1.09–3.15% DM; average 2.06% DM), while the nicotine content of 11 lines and 3 standards (Xanthi 2A, Nail, Xanthi 81) were 2.00% DM or higher (Table 3, Figure 3). On the other hand, nicotine content of tobacco grown in Evciler, Gümüşhacıköy and Bafra locations remained below the expectations of the sector. Nicotine content of tobacco can be increased to meet the market demand of 2.00–2.75% DM by agrigultural measures such as e.g., nitrogenous fertilization (26), by wider planting distances (39) and by topping (40). Since the assimilation of nitrogen takes place in the root system and the photosynthesis takes place in the leaves, organic compounds carrying carbon are synthesized only in the leaves, and nitrogenous compounds are synthesized in part in the root system. Therefore, various tobacco types rich in certain desired contents can be produced by modifying the external conditions (41). Topping on time and to a sufficient degree can cause the moving to a lower plant part of the photosynthesis products in the leaves, thus improving root growth and consequently facilitating assimilation of the nitrogenous compounds (41, 42).

Figure 3

Variation in nicotine content of 25 tobacco genotypes at four different locations.

Nicotine content, which is synthesized at the root tips, is closely related to root growth (41). Despite the expectation of high nicotine content in tobacco from the Evciler location (42) due to drier conditions compared to other locations, root and therefore plant growth were weak which hampered the increase in nicotine content.

Oriental tobaccos have a different flavor, lower nicotine levels compared to commercial flue-cured Virginia and air-cured Burley tobaccos and compared to Burley and bright tobaccos they also have lower tobacco specific nitrosamine (TSNA) levels. Therefore they are generally used in American blend type cigarettes by blending them with Virginia and Burley tobaccos (43). Precipitation and high humidity, especially during the harvest period, can delay maturation and leach down the resin and nicotine substances on the surface of the leaves. (44). Bafra is the location with the highest humidity.

If tobacco is cultivated in lighter soils, where the roots are well aerated and can take up the available water easily (under both limited nitrogen fertilization or under fertilized conditions), then tobacco with low levels of nitrogenous compounds is produced (41). The lower nicotine content of tobaccos grown in lowlands similar to the Bafra location has been attributed to a weaker root system as well as the lower fertility and less available nitrogen in lighter soils (8). Camas (45) studying regional tobaccos and their hybrids in Bafra stated that the lowest nicotine value of genotypes in the F1 generation was 0.47% DM (mean 1.04% DM) in Basma 192–23 varieties. The decrease in nicotine content was reported for the hybrids related to the Basma variety, and the results were considered promising to obtain tobacco with a low nicotine content. Camas et al. (46) and Esendal et al. (47,48,49) reported that nicotine content in tobacco grown in the Bafra region ranged from 0.3 to 2.1% DM. The nicotine contents obtained in this study were similar to those reported in these studies carried out in Bafra.

Sugars

Sugars, one of the most important components of the chemical structure of leaf tobacco, are primary metabolites (24) that contribute to the growth and development of tobacco plants. The reducing sugar composition is directly related to the taste and flavor of tobacco (30, 50, 51). The sugar content in different tobacco types is quite variable and depends primarily on the curing process (14) where longer curing periods cause a decrease in the sugar content (41, 42, 44).

Tobacco types are generally classified based on their curing methods. Reducing sugar in Oriental (sun-cured) and Virginia (flue-cured) tobaccos are relatively high (10–25% DM) compared to the reducing sugar content (< 2% DM) in Burley and Maryland (air-cured) tobaccos (53). Fructose content in this study was between 2.60 and 8.66% DM (Table 4). The fructose content of American blends, of which Virginia, Burley and Oriental tobacco are the main raw materials, is between 3.98 and 5.76% DM (52). Reducing sugar composition, together with phenolic compounds, resins and essential oils, determines the taste and flavor of tobacco (30, 50, 51). The reducing sugar content of an American blend is between 4.92 and 8.28% DM (52).

Glucose, fructose and reducing sugar content of 25 genotypes in four locations.

Genotype Glucose (% DM) Fructose (% DM) Reducing sugar (glucose + fructose) (% DM)



Evciler Kara. Güm. Bafra Mean Evciler Kara. Güm. Bafra Mean Evciler Kara. Güm. Bafra Mean
ERB-5 1.95 k 3.63 hk 2.84 d 2.66 df 2.77 2.60 k 5.15 ei 5.02 4.74 4.38 4.54 i 8.79 ei 7.86 df 7.41 fg 7.15
ERB-6 4.65 bd 3.46 ik 3.96 a 2.66 df 3.68 5.58 cf 6.15 cd 5.63 4.99 5.59 10.23 cf 9.61 df 9.59 a 7.65 bg 9.27
ERB-7 3.03 ij 4.44 ef 4.01 a 3.18 be 3.66 4.92 eh 6.48 cd 5.16 5.42 5.50 7.95 g 10.92 c 9.17 ac 8.60 ac 9.16
ERB-9 3.75 fi 4.50 ef 3.02 d 2.65 df 3.48 4.94 eh 4.54 hj 5.11 4.95 4.88 8.69 fg 9.04 eh 8.13 bf 7.60 cg 8.36
ERB-11 2.83 j 3.55 ik 2.87 d 2.73 cf 2.99 5.04 dg 4.49 j 5.08 5.11 4.93 7.87 g 8.04 hi 7.95 cf 7.84 ag 7.93
ERB-12 1.16 l 4.29 eg 3.23 cd 3.07 bf 2.94 3.58 ij 5.95 ce 4.84 5.29 4.91 4.74 hi 10.24 cd 8.07 bf 8.36 ag 7.85
ERB-13 1.36 kl 4.08 fh 3.29 bd 2.72 cf 2.86 4.42 gi 5.73 cg 5.32 5.01 5.12 5.79 hi 9.81 de 8.61 ae 7.73 ag 7.99
ERB-14 4.26 cf 2.49 lm 3.88 ab 2.63 df 3.32 6.04 cd 3.59 k 5.34 4.81 4.95 10.30 ce 6.08 jl 9.21 ab 7.45 eg 8.26
ERB-15 4.08 df 4.10 fh 2.87 d 2.90 bf 3.49 5.79 ce 5.40 dh 5.00 4.98 5.29 9.87 df 9.50 dg 7.87 df 7.88 ag 8.78
ERB-16 1.67 kl 1.68 n 3.77 ac 3.08 bf 2.55 2.77 jk 3.38 k 5.30 4.75 4.05 4.44 i 5.06 l 9.07 ad 7.82 ag 6.60
ERB-17 4.60 be 3.58 hk 3.90 ab 2.90 bf 3.74 6.03 cd 4.83 hi 5.18 4.93 5.24 10.63 ce 8.41 gi 9.08 ad 7.83 ag 8.99
ERB-18 2.06 k 4.64 de 2.89 d 3.16 bf 3.19 4.02 hi 4.91 gi 5.00 5.09 4.76 6.08 h 9.55 dg 7.88 df 8.25 ag 7.94
ERB-19 4.13 df 3.85 gj 3.03 d 3.36 b 3.59 5.93 ce 5.23 ei 5.13 5.01 5.32 10.07 df 9.07 eh 8.16 bf 8.37 ag 8.92
ERB-21 6.80 a 3.93 gi 3.08 d 3.05 bf 4.21 7.95 a 5.06 fi 5.29 5.33 5.91 14.75 a 8.98 eh 8.37 bf 8.38 ag 10.12
ERB-23 4.06 df 1.79 n 2.96 d 3.93 a 3.19 5.23 dg 4.71 hi 5.16 4.78 4.97 9.29 eg 6.50 jk 8.12 bf 8.71 ab 8.16
ERB-25 3.88 eg 2.76 l 3.04 d 3.25 bc 3.23 5.37 dg 3.89 jk 5.15 5.24 4.91 9.26 eg 6.64 j 8.19 bf 8.49 af 8.15
ERB-26 4.97 bc 3.53 ik 2.68 d 3.43 ab 3.65 6.41 bc 5.82 cf 5.18 5.36 5.69 11.37 bd 9.35 dg 7.86 df 8.79 a 9.35
ERB-27 3.26 gj 2.66 l 2.86 d 3.32 b 3.02 4.63 fh 5.11 ei 5.04 5.24 5.00 7.89 g 7.77 i 7.90 df 8.56 ad 8.03
ERB-30 4.48 cf 5.63 c 2.98 d 3.44 ab 4.13 7.16 ab 8.66 a 5.02 5.07 6.48 11.64 bc 14.29 a 8.00 bf 8.51 ae 10.61
ERB-35 3.07 ij 2.10 mn 2.94 d 3.22 bd 2.83 5.16 dg 3.39 k 4.65 5.02 4.55 8.23 g 5.49 kl 7.59 ef 8.24 ag 7.39
ERB-38 3.17 hj 3.23 k 2.78 d 2.66 df 2.96 4.94 eh 5.40 dh 4.84 5.28 5.12 8.11 g 8.64 fi 7.62 ef 7.94 ag 8.08
Xan.2A 2.97 j 3.34 jk 2.85 d 2.65 df 2.95 5.02 eg 5.06 fi 5.01 4.92 5.00 7.99 g 8.40 gi 7.87 df 7.57 cg 7.96
Nail 5.19 b 8.88 a 2.81 d 2.68 cf 4.89 7.39 a 5.86 cf 4.78 4.70 5.68 12.58 b 14.74 a 7.59 ef 7.37 g 10.57
Canik 3.85 fh 6.89 b 2.89 d 2.58 f 4.05 5.49 cf 8.14 a 4.93 4.89 5.87 9.35 eg 15.03 a 7.82 df 7.48 dg 9.92
Xan.81 3.11 ij 5.03 d 2.66 d 2.60 ef 3.35 4.98 eh 7.25 b 4.65 5.08 5.49 8.09 g 12.28 b 7.31 f 7.68 bg 8.84
Mean 3.53 3.92 3.12 2.98 3.39 5.26 5.37 5.07 5.04 5.18 8.79 9.29 8.20 8.02 8.57
M. check 3.78 6.04 2.80 2.63 3.81 5.72 6.58 4.84 4.90 5.51 9.50 12.61 7.65 7.53 9.32
M. lines 3.49 3.52 3.19 3.05 3.31 5.17 5.14 5.12 5.07 5.12 8.65 8.66 8.30 8.11 8.43
LSD 0.05 0.64 0.46 0.59 0.49 0.85 0.74 0.62 0.56 0.35 0.99 1.04 0.89
CV (%) 11.09 7.17 11.51 10.03 9.91 8.39 7.46 6.80 9.40 6.47 7.74 6.80
Mean square and significance df
Genotype 5.09** 7.28** 0.54** 0.37** 4.76** 5.09** 0.15 0.12 18.76** 20.55** 1.05** 0.61* 24
Error 0.15 0.08 0.13 0.09 0.27 0.20 0.14 0.12 0.68 0.36 0.40 0.30 48

Values followed by different letters in each column are significantly different (*p<0.05) according to Duncan's test; LSD: least significant difference; CV: coefficent of variation; df: degree of freedom;

p<0.01; M. check: mean of checks; M. lines: mean of lines.

Late planting in the Gümüşhacıköy location caused a delay in the harvest and curing period and as a consequence the curing of the 2nd hand harvest was postponed to September. Decreasing temperatures in September made for a longer curing period. The temperatures in Erbaa after the 2nd hand harvest went up to 25.9 °C, but the temperatures in August/September, when the 2nd hand is cured in Gümüşhacıköy, were only at 22.3 and 20.8 °C. Similar conditions were found in the Bafra location where the 2nd hand harvest was harvested on September 4. There the temperature in the curing period was 21.2 °C (Table 2). The excessive relative humidity in addition to the lower ambient temperatures during the curing period caused a decrease of the sugar content. This was due to the prolonged curing period (41, 42). Bafra is the location with the highest relative humidity among the locations (Table 2). The lowest reducing sugar content of Bafra, compared to the other locations, can be attributed to those low temperatures and high relative humidity throughout the curing period. The reducing sugar content in high demand by leaf tobacco companies for the Basma type tobacco is between 8 and 13% DM (26), and indeed the average reducing sugar content in the study ranged from 6.60 to 10.57% DM (mean 8.58% DM) (Table 4, Figure 4).

Figure 4

Variation in reducing sugars (glucose + fructose) content of 25 tobacco genotypes at four different locations.

Phenolics

Secondary metabolites, in addition to nicotine and reducing sugars that directly affect the color, taste and flavor of tobacco and thus determine the characteristics of the smoking product, should be taken into consideration during the planning and production processes. The most important of these secondary metabolites are polyphenols and among them the most abundant in tobacco are chlorogenic acid and rutin (17). The chlorogenic acid values in this study were between 156.70 and 426.73 ppm (Table 5).

Chlorogenic acid, rutin and the sum of chlorogenic acid + rutin amounts of 25 genotypes in four locations.

Genotype Chlorogenic acid (ppm) Rutin (ppm) Chlorogenic acid + rutin (ppm)



Evciler Kara. Güm. Bafra Mean Evciler Kara. Güm. Bafra Mean Evciler Kara. Güm. Bafra Mean
ERB-5 248 ij 534 fg 83 kl 41 j 226.4 638 fk 615 ef 304 ce 144 hj 425.3 886 ij 1149 jl 387 gi 185 kl 651.7
ERB-6 490 a 773 d 164 c 74 gi 375.3 677 dh 857 ab 389 bc 189 ej 528.1 1167 bd 1630 bc 553 bc 263 gk 903.4
ERB-7 483 a 528 fh 75 l 136 cd 305.6 999 a 729 cd 309 ce 293 ac 582.6 1482 a 1256 hj 385 gi 429 bc 888.3
ERB-9 414 bc 385 j 95 j 63 hj 239.3 661 ej 464 h 358 bd 143 hj 406.6 1076 bg 848 o 453 dh 206 il 645.9
ERB-11 394 bd 449 i 109 i 116 df 267.1 1021 a 613 ef 334 be 303 ab 567.9 1415 a 1062 ln 443 eh 419 bd 835.0
ERB-12 343 dg 727 d 75 l 119 df 315.9 544 kl 669 df 249 e 217 dg 419.9 886 ij 1396 eg 324 i 337 eg 735.9
ERB-13 407 bc 531 fh 118 gh 235 a 322.8 694 dh 673 df 375 bd 305 ab 511.8 1101 be 1203 ik 494 bf 539 a 834.6
ERB-14 300 gh 474 gi 112 hi 99 eg 246.5 732 cf 646 df 297 de 230 cf 476.5 1032 eh 1120 km 409 fh 329 eg 723.0
ERB-15 322 eg 532 fg 125 fg 84 gi 265.9 642 fk 669 df 388 bc 253 be 488.1 964 fi 1201 ik 513 be 338 eg 754.1
ERB-16 309 gh 364 j 146 d 140 cd 239.7 647 fj 455 h 421 ab 253 be 444.2 956 gi 819 o 567 b 393 ce 683.9
ERB-17 374 ce 492 gi 185 b 61 ij 278.1 724 cg 597 fg 475 a 166 fj 490.6 1098 bf 1089 km 659 a 227 hk 768.6
ERB-18 367 cf 734 d 153 d 129 ce 345.8 594 hk 596 fg 365 bd 156 gj 427.7 962 gi 1330 fh 517 be 285 gi 773.6
ERB-19 261 hi 932 b 122 fg 94 fh 352.5 459 lm 649 df 331 ce 212 dh 412.9 719 k 1581 cd 453 dh 307 fh 765.4
ERB-21 444 ab 877 bc 204 a 74 gi 399.8 758 be 834 ab 479 a 171 fj 560.7 1203 b 1710 b 683 a 245 hk 960.5
ERB-23 310 gh 721 d 190 b 55 ij 319.3 754 ce 697 ce 487 a 141 ij 519.7 1064 cg 1417 ef 677 a 196 jl 838.9
ERB-25 408 bc 616.4 e 129 ef 158 bc 327.9 573 ik 514 gh 301 ce 295 ac 420.8 981 ei 1130 km 429 eh 454 bc 748.8
ERB-26 366 cf 1120 a 90 jk 130 ce 426.7 674 di 899 a 337 be 213 dh 530.8 1040 dg 2019 a 427 eh 343 dg 957.6
ERB-27 239 ik 857 c 83 kl 175 b 338.7 797 bc 638 df 333 be 308 ab 519.2 1036 dg 1495 de 416 fh 484 ab 857.9
ERB-30 326 eg 338 j 168 c 130 ce 240.6 851 b 780 bc 370 bd 325 a 581.8 1177 bc 1118 km 538 bd 455 bc 822.4
ERB-35 222 ik 180 k 83 kl 142 cd 156.7 565 jk 482 h 293 de 259 ae 399.9 787 jk 662 p 376 hi 401 ce 556.6
ERB-38 331 eg 332 j 171 c 80 gi 228.7 623 gk 643 df 287 de 194 di 436.9 954 gi 975 n 459 dh 274 gj 665.6
Xan.2A 193 k 467 hi 137 e 79 gi 219.2 706 cg 818 ab 291 de 199 di 503.9 899 hj 1286 gi 428 eh 279 gi 723.2
Nail 313 fh 752 d 104 i 54 ij 305.7 641 fk 629 ef 293 de 121 j 420.9 954 gi 1380 fg 397 gi 175 l 726.6
Canik 199 jk 582 ef 149 d 85 gi 253.9 417 m 662 df 343 bd 193 di 403.8 616 l 1244 hj 492 bf 278 gi 657.8
Xan.81 249 ij 386 j 119 gh 118 df 218.2 765 bd 644 df 355 bd 262 ad 506.7 1014 ei 1030 mn 475 cg 381 cf 724.9
Means 332.6 587.4 127.7 106.9 288.7 686.3 658.9 350.7 221.9 479.5 1018.9 1246.4 478.4 328.9 768.2
M. check 238.6 546.9 127.6 84.2 249.3 632.1 688.5 320.7 193.9 458.8 870.7 1235.5 448.2 278.1 708.1
M. lines 350.5 595.2 127.7 111.3 296.2 696.7 653.3 356.4 227.3 483.4 1047.2 1248.5 484.2 338.6 779.6
LSD 0.05 48.35 57.06 7.79 27.67 87.50 83.20 75.16 59.36 115.20 102.40 76.57 70.57
CV (%) 8.85 5.91 3.71 15.75 7.76 7.69 13.05 16.29 6.88 5.01 9.75 13.07
Mean square and significance df
Genotype ** ** ** ** ** ** ** ** ** ** ** ** 24
20764.6 146546.5 4337.5 5953.5 57835.8 40088.8 11817.3 11189.5 105917.9 266647.8 26430.2 29988.2
Error 867 1208 22.54 284 2840.52 2567 2096 1307 4921 3891 2175 1847 48

Values followed by different letters in each column are significantly different (*p < 0.05) according to Duncan's test; LSD: Least significant difference; CV: Coefficent of variation; df: degree of freedom;

p<0.01; M. check: Mean of checks; M. lines: Mean of lines.

The chlorogenic acid contents reported in various studies for Oriental tobacco were between 0.16 and 0.25% DM (54), 1.56 mg g−1 (15), 30.2 mg g−1 (55), 7.9 mg g−1 (56) and between 4.5 and 12.3 mg g−1 (57). Rutin contents in Oriental tobacco were reported as 1.28–1.52% DM (44), 0.49–0.98% DM (58), 0.55–0.66% DM (54), 4.240 mg g−1 (15), 2.8 mg g−1 (55), 6.2 mg g−1 (56), and 5.6–9.3 mg g−1 (57). The mean rutin content of lines in all locations was between 399.94 and 582.60 ppm, while the variation of each line at each location was between 121.05 and 1021.53 ppm (Table 5).

The content of chlorogenic acid + rutin, which constituted approximately 85% of the total polyphenols (44), was similar or lower compared to those reported in previous studies (15, 44, 54,55,56,57,58). The mean total contents of chlorogenic acid + rutin were 1018.95 ppm in Evciler, 1246.38 ppm in Karayaka, 478.42 ppm in Gümüşhacıköy and 328.96 ppm in Bafra (Table 5, Figure 5).

Figure 5

Variation in phenolics (chlorogenic acid + rutin) content of 25 tobacco genotypes at four different locations.

Chlorogenic acid + rutin content in Oriental tobacco was reported as 0.84–2.41% DM (58), 0.71–0.91% DM (54), 33.00 mg g−1 (55), 14.10 mg g−1 (56) and 10.10–21.60 mg g−1 (57). Xie et al. (15) reported chlorogenic acid + rutin content as 2.3% DM for flue-cured, 0.054% DM for Burley and 1.08% DM for Oriental tobaccos. Phenolic compounds directly contribute to the formation of leaf color, and oxidation of phenolic compounds causes mutations towards dark brown tones. The amount of chlorogenic acid increases rapidly, depending on the severity of the enzymatic activity on the first and second day during curing and subsequently decreases related to the browning reaction (44). The duration and regime of climatic events have a significant impact on the chemical structures of tobacco lines grown in the experimental locations. The prolongation of curing processes in Gümüşhacıköy and Bafra locations had a negative effect on the accumulation of phenolic compounds.

CONCLUSION

The tobacco plant has a high adaptability and demonstrates unique characteristics in the common growing regions. Central Black Sea region tobacco type is a type of oriental tobacco in high demand despite the higher cost of raw material compared to other tobacco types. In this study, 21 Basma type tobacco lines, which stand out in regard to some characteristics, have been tested in four locations where the tobacco production is common. With a number of checks, their performances were monitored and the effects of ecological differences on tobacco quality were determined. Chemical composition of tobacco leaves was significantly different among locations where ecological conditions, such as light, humidity, precipitation, altitude and temperature were different from each other. Information on chemical composition of tobacco leaves is important to develop new blends or to sustain the quality of existing blends. Despite additives such as casing ingredients, flavors, or humectants in blends, the main source of taste perceived by smokers is the tobacco. Detailed studies in Oriental tobaccos with a focus on the secondary metabolites, especially phenolics, which are affected by the environmental conditions will be useful.

Figure 1

Dendrogram showing the genetic affinity of tobacco genotypes.
Dendrogram showing the genetic affinity of tobacco genotypes.

Figure 2

Variation in quality grade index (% of A grades) values of 25 tobacco genotypes at four different locations.
Variation in quality grade index (% of A grades) values of 25 tobacco genotypes at four different locations.

Figure 3

Variation in nicotine content of 25 tobacco genotypes at four different locations.
Variation in nicotine content of 25 tobacco genotypes at four different locations.

Figure 4

Variation in reducing sugars (glucose + fructose) content of 25 tobacco genotypes at four different locations.
Variation in reducing sugars (glucose + fructose) content of 25 tobacco genotypes at four different locations.

Figure 5

Variation in phenolics (chlorogenic acid + rutin) content of 25 tobacco genotypes at four different locations.
Variation in phenolics (chlorogenic acid + rutin) content of 25 tobacco genotypes at four different locations.

Glucose, fructose and reducing sugar content of 25 genotypes in four locations.

Genotype Glucose (% DM) Fructose (% DM) Reducing sugar (glucose + fructose) (% DM)



Evciler Kara. Güm. Bafra Mean Evciler Kara. Güm. Bafra Mean Evciler Kara. Güm. Bafra Mean
ERB-5 1.95 k 3.63 hk 2.84 d 2.66 df 2.77 2.60 k 5.15 ei 5.02 4.74 4.38 4.54 i 8.79 ei 7.86 df 7.41 fg 7.15
ERB-6 4.65 bd 3.46 ik 3.96 a 2.66 df 3.68 5.58 cf 6.15 cd 5.63 4.99 5.59 10.23 cf 9.61 df 9.59 a 7.65 bg 9.27
ERB-7 3.03 ij 4.44 ef 4.01 a 3.18 be 3.66 4.92 eh 6.48 cd 5.16 5.42 5.50 7.95 g 10.92 c 9.17 ac 8.60 ac 9.16
ERB-9 3.75 fi 4.50 ef 3.02 d 2.65 df 3.48 4.94 eh 4.54 hj 5.11 4.95 4.88 8.69 fg 9.04 eh 8.13 bf 7.60 cg 8.36
ERB-11 2.83 j 3.55 ik 2.87 d 2.73 cf 2.99 5.04 dg 4.49 j 5.08 5.11 4.93 7.87 g 8.04 hi 7.95 cf 7.84 ag 7.93
ERB-12 1.16 l 4.29 eg 3.23 cd 3.07 bf 2.94 3.58 ij 5.95 ce 4.84 5.29 4.91 4.74 hi 10.24 cd 8.07 bf 8.36 ag 7.85
ERB-13 1.36 kl 4.08 fh 3.29 bd 2.72 cf 2.86 4.42 gi 5.73 cg 5.32 5.01 5.12 5.79 hi 9.81 de 8.61 ae 7.73 ag 7.99
ERB-14 4.26 cf 2.49 lm 3.88 ab 2.63 df 3.32 6.04 cd 3.59 k 5.34 4.81 4.95 10.30 ce 6.08 jl 9.21 ab 7.45 eg 8.26
ERB-15 4.08 df 4.10 fh 2.87 d 2.90 bf 3.49 5.79 ce 5.40 dh 5.00 4.98 5.29 9.87 df 9.50 dg 7.87 df 7.88 ag 8.78
ERB-16 1.67 kl 1.68 n 3.77 ac 3.08 bf 2.55 2.77 jk 3.38 k 5.30 4.75 4.05 4.44 i 5.06 l 9.07 ad 7.82 ag 6.60
ERB-17 4.60 be 3.58 hk 3.90 ab 2.90 bf 3.74 6.03 cd 4.83 hi 5.18 4.93 5.24 10.63 ce 8.41 gi 9.08 ad 7.83 ag 8.99
ERB-18 2.06 k 4.64 de 2.89 d 3.16 bf 3.19 4.02 hi 4.91 gi 5.00 5.09 4.76 6.08 h 9.55 dg 7.88 df 8.25 ag 7.94
ERB-19 4.13 df 3.85 gj 3.03 d 3.36 b 3.59 5.93 ce 5.23 ei 5.13 5.01 5.32 10.07 df 9.07 eh 8.16 bf 8.37 ag 8.92
ERB-21 6.80 a 3.93 gi 3.08 d 3.05 bf 4.21 7.95 a 5.06 fi 5.29 5.33 5.91 14.75 a 8.98 eh 8.37 bf 8.38 ag 10.12
ERB-23 4.06 df 1.79 n 2.96 d 3.93 a 3.19 5.23 dg 4.71 hi 5.16 4.78 4.97 9.29 eg 6.50 jk 8.12 bf 8.71 ab 8.16
ERB-25 3.88 eg 2.76 l 3.04 d 3.25 bc 3.23 5.37 dg 3.89 jk 5.15 5.24 4.91 9.26 eg 6.64 j 8.19 bf 8.49 af 8.15
ERB-26 4.97 bc 3.53 ik 2.68 d 3.43 ab 3.65 6.41 bc 5.82 cf 5.18 5.36 5.69 11.37 bd 9.35 dg 7.86 df 8.79 a 9.35
ERB-27 3.26 gj 2.66 l 2.86 d 3.32 b 3.02 4.63 fh 5.11 ei 5.04 5.24 5.00 7.89 g 7.77 i 7.90 df 8.56 ad 8.03
ERB-30 4.48 cf 5.63 c 2.98 d 3.44 ab 4.13 7.16 ab 8.66 a 5.02 5.07 6.48 11.64 bc 14.29 a 8.00 bf 8.51 ae 10.61
ERB-35 3.07 ij 2.10 mn 2.94 d 3.22 bd 2.83 5.16 dg 3.39 k 4.65 5.02 4.55 8.23 g 5.49 kl 7.59 ef 8.24 ag 7.39
ERB-38 3.17 hj 3.23 k 2.78 d 2.66 df 2.96 4.94 eh 5.40 dh 4.84 5.28 5.12 8.11 g 8.64 fi 7.62 ef 7.94 ag 8.08
Xan.2A 2.97 j 3.34 jk 2.85 d 2.65 df 2.95 5.02 eg 5.06 fi 5.01 4.92 5.00 7.99 g 8.40 gi 7.87 df 7.57 cg 7.96
Nail 5.19 b 8.88 a 2.81 d 2.68 cf 4.89 7.39 a 5.86 cf 4.78 4.70 5.68 12.58 b 14.74 a 7.59 ef 7.37 g 10.57
Canik 3.85 fh 6.89 b 2.89 d 2.58 f 4.05 5.49 cf 8.14 a 4.93 4.89 5.87 9.35 eg 15.03 a 7.82 df 7.48 dg 9.92
Xan.81 3.11 ij 5.03 d 2.66 d 2.60 ef 3.35 4.98 eh 7.25 b 4.65 5.08 5.49 8.09 g 12.28 b 7.31 f 7.68 bg 8.84
Mean 3.53 3.92 3.12 2.98 3.39 5.26 5.37 5.07 5.04 5.18 8.79 9.29 8.20 8.02 8.57
M. check 3.78 6.04 2.80 2.63 3.81 5.72 6.58 4.84 4.90 5.51 9.50 12.61 7.65 7.53 9.32
M. lines 3.49 3.52 3.19 3.05 3.31 5.17 5.14 5.12 5.07 5.12 8.65 8.66 8.30 8.11 8.43
LSD 0.05 0.64 0.46 0.59 0.49 0.85 0.74 0.62 0.56 0.35 0.99 1.04 0.89
CV (%) 11.09 7.17 11.51 10.03 9.91 8.39 7.46 6.80 9.40 6.47 7.74 6.80
Mean square and significance df
Genotype 5.09** 7.28** 0.54** 0.37** 4.76** 5.09** 0.15 0.12 18.76** 20.55** 1.05** 0.61* 24
Error 0.15 0.08 0.13 0.09 0.27 0.20 0.14 0.12 0.68 0.36 0.40 0.30 48

Monthly climate data during vegetative growth in the locations.

Months Erbaa Gümüşhacıköy Bafra



LTA. 2017 D. LTA. 2017 D. LTA. 2017 D.
April °C 14.2 12.4 −1.8 11.0 10.0 −1.0 11.2 9.9 −1.3
mm 55.4 45.2 −10.2 56.9 43.6 −13.3 57.7 63.0 5.3
% 58.6 65.2 6.6 58.7 62.9 4.2 79.0 85.8 6.8
May °C 18.1 17.0 −1.1 14.4 14.4 0 15.5 15.0 −0.5
mm 62.2 50.5 −11.7 75.5 70.9 −4.6 47.2 53.6 6.4
% 60.7 71.6 10.9 59.6 69.5 9.9 79.4 87.6 8.2
June °C 21.6 21.4 −0.2 18.2 18.4 0.2 20.1 20.3 0.2
mm 48.4 94.5 46.1 74.9 87.3 12.4 34.6 45.9 11.3
% 58.1 73.6 15.5 58.9 79.2 20.3 74.9 84.7 9.8
July °C 23.9 20.8 −3.1 20.7 21.3 0.6 22.8 23.5 0.7
mm 24.6 1.2 −23.4 26.6 8.4 −18.2 31.0 0 −31
% 55.4 74.2 18.8 53.3 55.5 2.2 73.1 95.2 22.1
August °C 23.8 25.9 2.1 21.1 22.3 1.2 22.9 24.3 1.4
mm 9.9 1.0 −8.9 11.3 9.2 −2.1 47.3 24.8 −22.5
% 55.6 65.8 10.2 50.9 67.7 16.8 74.5 75.2 0.7
September °C 20.5 22.2 1.7 17.4 20.8 3.4 19.5 21.2 1.7
mm 16.1 2.9 −13.2 13.0 13.2 0.2 59.7 13.0 −46.7
% 57.9 61.6 3.7 53.9 51.7 −2.2 76.8 75.2 −1.6
October °C 15.4 13.9 −1.5 12.3 12.2 −0.1 15.4 15.4 0
mm 41.7 26.9 −14.8 45.4 23.9 −21.5 96.8 21.8 −75
% 63.2 77.0 13.8 61.4 64.5 3.1 78.7 72.0 −6.7
Mean temperature (°C) 19.6 19.1 −0.6 16.4 17.1 0.6 18.2 18.5 0.3
Total precipitation (mm) 258.3 222.2 −36.1 303.6 256.5 −47.1 374.3 222.1 −152.2
Mean relative humidity (%) 58.5 69.9 11.4 56.7 64.4 7.8 76.6 82.2 5.6

Soil analysis results of the locations.

Properties Locations

Evciler Karayaka Gümüşhacıköy Bafra
P2O5 (kg ha−1) 0.51 low 0.62 moderate 0.48 low 0.34 low
K2O (kg ha−1) 16.97 high 17.53 high 15.68 high 13.72 high
Lime (%) 10.2 moderately calcareous 2.39 calcareous 5.17 moderately calcareous 12.73 moderately calcareous
Org. Mat. (%) 95 very low 143 low 2.36 moderate 1.76 low
pH 7.99 slightly alkaline 7.81 slightly alkaline 7.98 slightly alkaline 7.61 slightly alkaline
EC (dS m−1) 0.25 very low 0.13 very low 1.12 very low 0.72 very low
Texture clay loam sandy loam sandy loam sandy loam

Chlorogenic acid, rutin and the sum of chlorogenic acid + rutin amounts of 25 genotypes in four locations.

Genotype Chlorogenic acid (ppm) Rutin (ppm) Chlorogenic acid + rutin (ppm)



Evciler Kara. Güm. Bafra Mean Evciler Kara. Güm. Bafra Mean Evciler Kara. Güm. Bafra Mean
ERB-5 248 ij 534 fg 83 kl 41 j 226.4 638 fk 615 ef 304 ce 144 hj 425.3 886 ij 1149 jl 387 gi 185 kl 651.7
ERB-6 490 a 773 d 164 c 74 gi 375.3 677 dh 857 ab 389 bc 189 ej 528.1 1167 bd 1630 bc 553 bc 263 gk 903.4
ERB-7 483 a 528 fh 75 l 136 cd 305.6 999 a 729 cd 309 ce 293 ac 582.6 1482 a 1256 hj 385 gi 429 bc 888.3
ERB-9 414 bc 385 j 95 j 63 hj 239.3 661 ej 464 h 358 bd 143 hj 406.6 1076 bg 848 o 453 dh 206 il 645.9
ERB-11 394 bd 449 i 109 i 116 df 267.1 1021 a 613 ef 334 be 303 ab 567.9 1415 a 1062 ln 443 eh 419 bd 835.0
ERB-12 343 dg 727 d 75 l 119 df 315.9 544 kl 669 df 249 e 217 dg 419.9 886 ij 1396 eg 324 i 337 eg 735.9
ERB-13 407 bc 531 fh 118 gh 235 a 322.8 694 dh 673 df 375 bd 305 ab 511.8 1101 be 1203 ik 494 bf 539 a 834.6
ERB-14 300 gh 474 gi 112 hi 99 eg 246.5 732 cf 646 df 297 de 230 cf 476.5 1032 eh 1120 km 409 fh 329 eg 723.0
ERB-15 322 eg 532 fg 125 fg 84 gi 265.9 642 fk 669 df 388 bc 253 be 488.1 964 fi 1201 ik 513 be 338 eg 754.1
ERB-16 309 gh 364 j 146 d 140 cd 239.7 647 fj 455 h 421 ab 253 be 444.2 956 gi 819 o 567 b 393 ce 683.9
ERB-17 374 ce 492 gi 185 b 61 ij 278.1 724 cg 597 fg 475 a 166 fj 490.6 1098 bf 1089 km 659 a 227 hk 768.6
ERB-18 367 cf 734 d 153 d 129 ce 345.8 594 hk 596 fg 365 bd 156 gj 427.7 962 gi 1330 fh 517 be 285 gi 773.6
ERB-19 261 hi 932 b 122 fg 94 fh 352.5 459 lm 649 df 331 ce 212 dh 412.9 719 k 1581 cd 453 dh 307 fh 765.4
ERB-21 444 ab 877 bc 204 a 74 gi 399.8 758 be 834 ab 479 a 171 fj 560.7 1203 b 1710 b 683 a 245 hk 960.5
ERB-23 310 gh 721 d 190 b 55 ij 319.3 754 ce 697 ce 487 a 141 ij 519.7 1064 cg 1417 ef 677 a 196 jl 838.9
ERB-25 408 bc 616.4 e 129 ef 158 bc 327.9 573 ik 514 gh 301 ce 295 ac 420.8 981 ei 1130 km 429 eh 454 bc 748.8
ERB-26 366 cf 1120 a 90 jk 130 ce 426.7 674 di 899 a 337 be 213 dh 530.8 1040 dg 2019 a 427 eh 343 dg 957.6
ERB-27 239 ik 857 c 83 kl 175 b 338.7 797 bc 638 df 333 be 308 ab 519.2 1036 dg 1495 de 416 fh 484 ab 857.9
ERB-30 326 eg 338 j 168 c 130 ce 240.6 851 b 780 bc 370 bd 325 a 581.8 1177 bc 1118 km 538 bd 455 bc 822.4
ERB-35 222 ik 180 k 83 kl 142 cd 156.7 565 jk 482 h 293 de 259 ae 399.9 787 jk 662 p 376 hi 401 ce 556.6
ERB-38 331 eg 332 j 171 c 80 gi 228.7 623 gk 643 df 287 de 194 di 436.9 954 gi 975 n 459 dh 274 gj 665.6
Xan.2A 193 k 467 hi 137 e 79 gi 219.2 706 cg 818 ab 291 de 199 di 503.9 899 hj 1286 gi 428 eh 279 gi 723.2
Nail 313 fh 752 d 104 i 54 ij 305.7 641 fk 629 ef 293 de 121 j 420.9 954 gi 1380 fg 397 gi 175 l 726.6
Canik 199 jk 582 ef 149 d 85 gi 253.9 417 m 662 df 343 bd 193 di 403.8 616 l 1244 hj 492 bf 278 gi 657.8
Xan.81 249 ij 386 j 119 gh 118 df 218.2 765 bd 644 df 355 bd 262 ad 506.7 1014 ei 1030 mn 475 cg 381 cf 724.9
Means 332.6 587.4 127.7 106.9 288.7 686.3 658.9 350.7 221.9 479.5 1018.9 1246.4 478.4 328.9 768.2
M. check 238.6 546.9 127.6 84.2 249.3 632.1 688.5 320.7 193.9 458.8 870.7 1235.5 448.2 278.1 708.1
M. lines 350.5 595.2 127.7 111.3 296.2 696.7 653.3 356.4 227.3 483.4 1047.2 1248.5 484.2 338.6 779.6
LSD 0.05 48.35 57.06 7.79 27.67 87.50 83.20 75.16 59.36 115.20 102.40 76.57 70.57
CV (%) 8.85 5.91 3.71 15.75 7.76 7.69 13.05 16.29 6.88 5.01 9.75 13.07
Mean square and significance df
Genotype ** ** ** ** ** ** ** ** ** ** ** ** 24
20764.6 146546.5 4337.5 5953.5 57835.8 40088.8 11817.3 11189.5 105917.9 266647.8 26430.2 29988.2
Error 867 1208 22.54 284 2840.52 2567 2096 1307 4921 3891 2175 1847 48

Quality grade index and nicotine contents of 25 genotypes in four locations.

Genotypes Quality grade index (%) Nicotine (% DM)


Evciler Karayaka G.Hacıköy Bafra Mean Evciler Karayaka G.Hacıköy Bafra Mean
ERB-5 96.09 ad 74.96 ae 65.05 fi 69.27 a 76.34 0.71 fg 1.81 dh 1.02 bd 0.34 fg 0.97
ERB-6 90.97 ce 75.77 ad 71.75 dg 50.87 ae 72.34 1.02 af 1.28 gh 1.03 bd 0.32 fg 0.91
ERB-7 96.77 ac 79.93 ab 73.07 df 62.36 ac 78.03 1.16 ad 2.02 bg 0.59 hi 0.52 be 1.07
ERB-9 80.06 gh 64.64 bg 43.63 k 39.37 df 56.92 0.87 cg 2.04 bg 0.78 di 0.31 g 1.00
ERB-11 73.20 i 48.92 gh 69.00 eh 39.18 df 57.57 0.88 bg 1.49 fh 0.91 cg 0.46 bg 0.93
ERB-12 96.87 ac 68.19 af 65.13 fi 56.00 ad 71.55 0.82 dg 2.69 ac 0.63 gi 0.61 ac 1.19
ERB-13 98.55 ab 69.60 af 79.93 ad 66.52 ab 78.65 1.32 a 2.59 ad 0.86 ch 0.68 a 1.36
ERB-14 91.77 ce 61.81 cg 58.53 hj 57.65 ad 67.44 1.17 ac 2.58 ad 0.68 fi 0.48 bg 1.23
ERB-15 100.00 a 65.66 bf 88.26 ab 57.10 ad 77.75 0.96 bf 2.30 be 0.92 cg 0.57 ad 1.19
ERB-16 89.22 ef 61.55 cg 76.78 ce 40.47 df 67.01 0.77 eg 1.09 h 1.14 ac 0.41 dg 0.85
ERB-17 85.31 fg 61.30 cg 47.71 k 46.47 be 60.20 1.16 ad 1.69 eh 0.96 cf 0.41 dg 1.06
ERB-18 76.55 hi 61.04 dg 53.04 jk 32.97 ef 55.90 0.83 cg 2.01 cg 1.13 ac 0.47 bg 1.11
ERB-19 90.49 df 83.53 a 87.37 ac 57.41 ad 79.70 0.83 cg 2.07 bf 0.86 ch 0.49 bf 1.06
ERB-21 80.50 gh 65.54 bf 85.38 ac 59.05 ad 72.62 1.05 ae 1.57 eh 0.93 cg 0.36 eg 0.98
ERB-23 95.12 ad 65.72 bf 64.96 fi 67.54 a 73.34 1.14 ad 2.70 ac 0.94 cf 0.40 dg 1.29
ERB-25 76.35 hi 57.89 fg 60.80 hj 44.09 ce 59.78 0.99 fg 1.33 fh 0.92 cg 0.46 bg 0.92
ERB-26 94.35 ae 78.73 ab 57.80 ij 48.99 ae 69.96 0.95 bf 2.52 ad 0.74 di 0.47 bg 1.17
ERB-27 76.07 hi 40.33 h 46.33 k 24.17 f 46.72 0.85 cg 1.53 eh 0.73 di 0.46 bg 0.89
ERB-30 93.33 be 70.14 af 89.89 a 48.87 ae 75.56 1.06 ae 1.51 fh 1.35 a 0.45 bg 1.09
ERB-35 81.44 gh 79.14 ab 85.21 ac 55.77 ad 75.39 0.70 fg 1.99 cg 0.53 i 0.60 ac 0.96
ERB-38 96.65 ac 77.77 ac 84.23 ac 53.72 ad 78.09 1.03 af 2.66 ac 0.86 ch 0.44 cg 1.25
Xanthi 2A 96.84 ac 82.19 a 78.73 be 50.53 ae 77.07 0.99 af 2.81 ab 0.99 be 0.57 ad 1.34
Nail 94.62 ae 68.16 af 62.19 gj 61.33 ac 71.58 1.08 ae 2.58 ad 0.78 di 0.40 dg 1.21
Canik 190–5 94.75 ae 58.94 eg 68.70 eh 59.43 ad 70.45 0.54 g 1.40 fh 1.25 ab 0.42 dg 0.90
Xanthi 81 95.81 ad 79.72 ab 76.68 ce 58.46 ad 77.67 1.22 ab 3.15 a 0.70 ei 0.62 ab 1.42
Mean 89.67 68.05 69.61 52.30 69.91 0.96 2.06 0.89 0.47 1.09
M. checks 95.50 72.26 71.57 57.44 74.19 0.96 2.48 0.93 0.50 1.22
M. lines 88.56 67.25 69.23 51.32 69.09 0.97 1.97 0.88 0.46 1.07
LSD0.05 4.97 13.77 9.30 17.06 0.28 0.66 0.25 0.14
CV (%) 3.37 12.32 8.13 19.87 17.97 19.65 17.14 18.80
Mean square and significance df
Genotype 207.26** 336.07** 564.63** 364.88** 0.10** 0.98** 0.12** 0.03** 24
Error 9.15 70.31 32.09 107.99 0.03 0.16 0.02 0.01 48

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