Tobacco-specific nitrosamines (TSNAs) are harmful carcinogens, with nitrate as a precursor of their formation. Nitrate content is considerably higher in burley tobacco than in flue-cured tobacco, but little has been reported on the differences between types of nitrate accumulation during development. We explored nitrate accumulation prior to harvest and examined the effects of regulatory substances aimed at decreasing nitrate and TSNA accumulation. In growth experiments, nitrate accumulation in burley and flue-cured tobacco initially increased but then declined with the highest nitrate content observed during a fast-growth period. When treating tobacco crops with molybdenum (Mo) during fast growth, nitrate reductase activity in burley tobacco increased significantly, but the NO3-N content decreased. These treatments also yielded significant reductions in NO3-N and TSNA contents. Therefore, we suggest that treatment with Mo during the fast-growth period and a Mo-Gfo (Mo-glufosinate) combination at the maturity stage is an effective strategy for decreasing nitrate and TSNAs during cultivation.
Eight types of tobacco-specific nitrosamines (TSNAs) are present in tobacco with the majority known to cause malignant tumors in mice, rats, and hamsters [
Nitrate (
Many factors such as nitrogen management, soil fertility, tobacco types and varieties, and cultivation conditions are related to nitrate accumulation [
The objective of the present study was to explore characteristics of nitrate accumulation in both burley and flue-cured tobacco and compare the differences between types in nitrate reductase activity (NRA) and NRA/nitrogen application (NA) to develop strategies for their regulation during cultivation. A field experiment using chemical regulation was conducted to decrease nitrate and TSNA concentrations in flue-cured tobacco, and the effects of spraying regulated substances on burley varieties TN86 and TN90 were analyzed to determine an effective method for reducing nitrate and TSNA concentrations in burley tobacco. The effects of spraying Mo during the fast-growth period and at the maturity stage and of spraying Mo and Gfo together at maturity on NRA, GSA, ammonia volatilization rate (AVR), soluble protein content (SPRO), TSNAs, and TSNA precursors were determined.
Field and pot experiments were conducted in 2015 in Henan, China (33°15′52.14′′N, 112°55′28.51′′E), using two tobacco types to explore nitrate accumulation in tobacco. Two burley tobacco genotypes, TN86 and KT204, and two cultivars of flue-cured tobacco, honghuadajinyuan (HD) and yunyan 87 (Y87), were used. Mean temperature and precipitation in this region were 24.1°C and 510 mm, respectively, during tobacco cultivation season (from May to September each year).
The soil in the field was mainly yellow loamy soil. Soil properties were tested at a depth of 0–30 cm before transplanting and consisted of an organic matter content of 13.55 g kg−1, available N of 55.01 mg kg−1, available K of 120.63 mg kg−1, and available P of 18.21 mg kg−1, and a pH of 7.13. Nitrogen application was 45 kg ha−2 and 180 kg ha−2 for flue-cured tobacco and burley tobacco, respectively. Plants were placed at a density of one plant per 0.605 m2 (column and line spacing per plant: 0.55 × 1.10 m, resp.) in field experiments. Tobacco seedlings were transplanted to the field on May 1, 2015. Burley tobacco was cut once, on July 17, 2015, and flue-cured tobacco was picked 3–5 times beginning on July 12 at 7–9-day intervals. Experimental treatments consisted of a randomized block design with three replicates. Leaf biomass was collected at 30, 45, 60, and 75 days after transplantation (DAT) in field-grown plants, with the final samples picked just prior to harvest. Fresh leaves were fixed for 20 min at 105°C and then dried for 48 h at 60°C. NRA and NO3-N contents in leaf were determined at 30, 45, 60, and 75 DAT in field.
For the pot experiments, the soil was mainly yellow loamy soil. Soil was tested at a depth of 0–30 cm before transplanting and was similar to that of the field experiments with an organic matter content of 13.55 g kg−1, available N of 55.10 mg kg−1, available K of 120.76 mg kg−1, available P of 18.20 mg kg−1, and a pH of 7.13. Nitrogen application was 45 kg ha−2 and 180 kg ha−2 for flue-cured tobacco and burley tobacco, respectively. Plants were placed at a density of one plant per 0.605 m2 (column and line spacing per plant: 0.55 × 1.10 m, resp.) and transplanted to pots with a 50 cm outer diameter, 42.5 cm inner diameter, and 33 cm height and were buried to a depth of 20–25 cm on May 15, 2015. Leaf biomass was collected after transplantation at 15, 30, 45, and 60 DAT, with the final samples picked just before harvest. NRA and NO3-N contents in the leaves were determined at 15, 30, 45, and 60 DAT.
Nitrate regulation experiments were conducted in 2014 (Yunnan, China, 25°21′17.37′′N, 100°28′6.75′′E) and 2015 (Henan, China, 33°15′52.14′′N, 112°55′28.51′′E) using flue-cured tobacco (HD).
The soil in which the plants were grown was mainly paddy soil with a mean temperature and precipitation of 18.5°C and 625 mm, respectively, during tobacco cultivation season from May to September each year. Soil properties were tested at a depth of 0–20 cm prior to transplantation and had an organic matter content of 22.4 g kg−1, available N of 120.01 mg kg−1, available K of 154.63 mg kg−1, P of 28.4 mg kg−1, and pH of 6.48.
Field soil was mainly yellow loamy soil. Annual mean temperature and precipitation in this region were 24.1°C and 510 mm, respectively, during tobacco cultivation season from May to September each year. Soil properties were tested at a depth of 0–30 cm before transplanting and had an organic matter content of 13.55 g kg−1, available N of 55.01 mg kg−1, available K of 120.63 mg kg−1, and available P of 18.21 mg kg−1, and pH of 7.13. Nitrogen applications were 75 kg ha−2 and 45 kg ha−2 in 2014 and 2015, respectively. Tobacco seedlings were transplanted on May 7, 2014, and May 1, 2015. Spraying during fast-growth periods or at the stage of maturity was carried out on June 17 and July 15, 2014, and June 11 and July 10, 2015, respectively. TSNAs, NO3-N, NO2-N, and alkaloids in the tobacco were determined after curing. Field management was carried out according to conventional practice.
The following treatments were applied: A control treatment, wherein water only was sprayed during the fast-growth and maturity stages (CK) Sodium molybdate sprayed during the fast-growth period (FG-Mo) Sodium molybdate sprayed during the fast-growth period and Gfo sprayed at the stage of maturity (M-Gfo) Sodium molybdate sprayed during the fast-growth period and sodium molybdate combined with Gfo sprayed at the maturity stage (M-Mo + Gfo).
Sodium molybdate (Mo) and Gfo doses were determined in preliminary tests, and 10 mg L−1 Gfo (v/v) and 0.5% (m/m) Mo were screened out to spray in field experiments. The dose of Gfo sprayed on tobacco (0.01 kg hm−2) was much lower than its use as a herbicide during agriculture cultivation (0.40 kg hm−2 used to control annual weeds and 1-2 kg hm−2 used to control perennial weeds) [
Nitrate regulation experiments on burley tobacco (TN86 and KT204 varieties) were conducted in 2015 in Henan, China (33°15′52.14′′N, 112°55′28.51′′E). Soil conditions, treatments, and management were as described in experiment 2. NRA and NO3-N content were determined five days after spraying during the fast-growth period. NRA, GSA, NO3-N, and SPRO were determined at the seventh day after spraying, and ammonia volatilization was measured for one full 24 h period from 08:00 to 08:00 on the seventh day after spraying at the stage of maturity. AVR was calculated as the ratio of the amount of ammonia volatilization over time. TSNAs, NO3-N, NO2-N, and alkaloids in the tobacco were determined after curing.
The length of the various stages of tobacco development is as follows [
Soil pH was determined in 1 : 2.5 (v/v) soil/water suspension, organic matter content was determined using the potassium bichromate titrimetric method, available nitrogen was measured by using the alkaline hydrolysis diffusion method, available potassium was measured using the neutral ammonium acetate extraction method, and available phosphorus was determined using alkaline sodium bicarbonate as the extractant in a 20 : 1 ratio [
Tobacco leaves were sampled at 10:00–11:00 a.m. on sunny days. Samples were frozen and fresh leaves without veins were cut into 2 × 5 mm pieces before measurement. NRA was measured based on the method described by Li [
Tobacco samples were lyophilized, ground, and sieved through a 0.25 mm screen prior to measurement. TN was determined using methods modified from the Chinese Tobacco Industry standard (YC/T 161,159-2002). Samples of 0.1 g powder mixture containing 0.1 g CuSO4 and 1 g K2SO4 were mixed with 5 mL of concentrated H2SO4 (98.3% m/m) in a 50 mL digestion tube and held for 1-2 h at room temperature. Samples were then warmed to 150°C for 30 min, 250°C for 30 min, and 370°C for 2 h in a furnace (DS53-380, CIF, USA). After cooling, approximately 10 mL deionized water was added, and samples were shaken thoroughly. Sample mixtures were cooled for 1-2 h, and water was added to maintain the overall volume of the samples. The mixtures were then cooled for 1 h and filtered. TN in the supernatant was determined using flow-injection-analysis (AA3, Bran + Luebbe, Germany).
NO3-N and NO2-N were quantified according to Crutchfield and Grove [
Comparisons were made using analyses of variance (ANOVAs) and least significant differences for NRA, GSA, AVR, NOX, alkaloids, and TSNAs with
In field and pot experiments, nitrate content in both burley tobacco and flue-cured tobacco increased over the period of development and presented a trend of “rise-fall” prior to harvest (Figure
Difference between burley tobacco and flue-cured tobacco in NRA, NRA/NA, and NO3-N content of leaves. Burley tobacco varieties were KT204 and TN86, and flue-cured tobacco varieties were HD and Y87. NA: nitrogen application (HD and Y87: 45 kg ha−2, KT204 and TN86: 180 kg ha−2). NRA: nitrate reductase activity. Error bars indicate standard error of the means (
In general, the amount of nitrogen fertilizers used on burley tobacco was almost 3–5 times higher than that used on flue-cured tobacco, but the yield was not significantly different between them [
LDM and DM were used to evaluate whether plants were growing well and to predict yield in tobacco cultivation [
ANOVA results of the effects of chemical regulation, year, and tobacco variety and their interactions on LDM and DM before harvest.
Year | Treatment | LDM (g/plant) | DM (g/plant) | Variety | Treatment | LDM (g/plant) | DM (g/plant) |
---|---|---|---|---|---|---|---|
2014 | CK | | | KT204 | CK | | |
FG-Mo | | | FG-Mo | | | ||
M-Gfo | | | M-Gfo | | | ||
M-Mo + Gfo | | | M-Mo + Gfo | | | ||
| |||||||
2015 | CK | | | TN86 | CK | | |
FG-Mo | | | FG-Mo | | | ||
M-Gfo | | | M-Gfo | | | ||
M-Mo + Gfo | | | M-Mo + Gfo | | | ||
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Year (Y) | | | Variety (V) | | | ||
| |||||||
Treatment (T) | | | Treatment (T) | | | ||
| |||||||
Year (Y) × treatment (T) | | | Variety (V) × treatment (T) | | |
Different letters within the same column indicate significant differences among treatments at
NRA and NO3-N content in both TN86 and KT204 exhibited increasing trends (Figure
Changes in the NRA and NO3-N content of burley tobacco under Mo treatment during fast-growth period. NRA: nitrate reductase activity. NRA in tobacco was determined at 6 h and on days 1–5 after spraying. Error bars indicate standard error of means (
Composition of tobacco at the stage of maturity is significantly indicative of the components of cured tobacco, and improving chemical composition during this stage is useful in enhancing tobacco quality [
Effects of Mo and Gfo treatments on NRA, AVR, GSA, and SPRO in burley tobacco. Error bars represent standard error (
NO3-N, NO2-N, and alkaloids are precursors of TSNAs, and decreasing precursors is effective in reducing TSNA formation in tobacco. Sufficient NO3-N content can greatly promote TSNA formation during tobacco storage, and reducing NO3-N accumulation is key in decreasing TSNA formation [
Effects of treatments on total nitrogen content, NO3-N content, NO2-N content, alkaloid content, and NO3-N/TN in burley tobacco. NO3-N/TN: ratio of NO3-N and total nitrogen content (TN). Error bars represent standard error (
Auxin, naphthylacetic acid, salicylic acid, and malonic acid have been previously applied to decrease TSNA formation, but these may affect tobacco development and growth, yield, or quality [
ANOVA comparison of the effects of regulatory treatments, year, and tobacco variety and their interactions on TN, NO3-N, NO2-N, alkaloid, and TSNA concentrations in tobacco.
Types | Effect | TN | NO3-N | NO2-N | Alkaloid | TSNAs | DF |
---|---|---|---|---|---|---|---|
Flue-cured tobacco | Year (Y) | 0.05 | 0.20 | 0.01 | 1.60 | 0.04 | 1 |
Treatment (T) | 9.01 | 15.33 | 0.21 | 4.65 | 7.19 | 3 | |
Year (Y) × Treatment (T) | 8.54 | 55.61 | 13.71 | 5.73 | 30.52 | 7 | |
| |||||||
Burley tobacco | Variety (V) | 0.97 | 0.18 | 11.08 | 0.02 | 1.18 | 1 |
Treatment (T) | 3.59 | 19.34 | 0.97 | 0.28 | 18.68 | 3 | |
Variety (V) × Treatment (T) | 5.66 | 275.57 | 28.41 | 13.14 | 274.77 | 7 |
As can be seen in Figure
Effects of chemical regulation on NNN, NAB, NAT, NNK, and total TSNA concentration in flue-cured tobacco. Error bars indicate standard error (
TSNA accumulation in burley tobacco was much higher than in flue-cured tobacco. However, effects of regulatory treatment were more pronounced in burley tobacco. Within burley varieties, the TSNA concentrations in KT204 were higher than that in TN86 (Figures
Effects of chemical regulation on NNN, NAB, NAT, NNK, and total TSNA concentration in burley tobacco varieties, TN86 and KT204. Error bars represent standard error (
Linear relationships between TSNAs, alkaloids, and NO3-N were significantly different (Figures
Correlation analysis between TSNAs, alkaloid, NO2-N, and NO3-N in tobacco. Symbol
Mechanisms for decreasing nitrate and TSNA concentrations in tobacco by spraying regulating chemicals. Gfo: glufosinate, NR: nitrate reductase, NRA: nitrate reductase activity, NiR: nitrite reductase, GS: glutamine synthetase, GSA: glutamine synthetase activity, Gln: glutamine, Glu: glutamate, and OG: oxaloacetate. After spraying Mo on tobacco during the fast-growth period, nitrate significantly decreased while NRA and soluble protein content increased. These decreased the amount of nitrate storage and promoted tobacco development during the fast-growth period. After spraying Mo during the fast-growth stage and spraying Mo and Gfo at the stage of maturity, NRA increased and GSA decreased in tobacco, which can significantly reduce nitrate accumulation and TSNA formation by nitrogen loss due to ammonia volatilization.
Nitrate was higher in burley tobacco than in flue-cured tobacco, with both types showing peak nitrate content during the fast-growth period. Under Mo treatment at the stage of maturity to avoid nitrate accumulation, NRA, LDM, and DM in tobacco leaves increased. Spraying Mo in combination with Gfo at the stage of maturity led to increased NRA and lower GSA in tobacco, which could help decrease nitrate and nitrite content by increasing nitrogen loss via ammonia volatilization. In summary, spraying Mo during fast growth and spraying Mo with Gfo at the stage of maturity were effective in reducing the formation of TSNAs.
The authors declare that there are no conflicts of interest regarding the publication of this paper.
The authors thank Editage (