| lant nitrate reductase activity (NRA) decreased rapidly after harvest and was near zero after three days of curing and did not result in detectable nitrite accumulation. In vitro NRA, most likely from microbial activity and not plant NRA, increased after three weeks of curing which corresponded to significant leaf moisture loss, degradation of the cell membrane, and significant nitrite accumulation. Midrib nitrite increased after three weeks of curing. Tobacco-specific nitrosamine (TSNA) increased rapidly following the accumulation of nitrite. There was always a significant and positive correlation between in vitro NRA and nitrite accumulation, between nitrite and TSNA accumulation regardless of year, cultivar or curing conditions. Thus the formation of nitrite by microbial NRA is the limiting step for TSNA accumulation during air-curing tobacco.;A seed grafting method, grafting nitrate reductase (NR) deficient mutants of Nicotiana. plumbaginifolia onto Nicotiana. tabacum plants (W-38), was developed and enough NR mutant plants were produced in the greenhouse for a curing study. The grafted NR mutant plants did accumulate nitrite and TSNA during air-curing. Since these leaves did not have any endogenous NRA, the NO;Greenhouse grown tobacco can significantly accumulate TSNA during air-curing by increased fertilization, leaf contamination with a soil slurry, and high curing humidity in the curing structure. The increased TSNA in the soil slurry treated tobacco was a direct result of increased nitrite formation by in vitro NRA at the end-of-yellowing (EOY).;The primed leaf treatments significantly increased leaf moisture loss, yielded at least 50% less nitrite formation, and consequently resulted in over 50% to 70% less TSNA accumulation compared to the stalk cured control. Thus, the quicker leaf moisture is lost, less nitrite and TSNA formation occur. TSNA accumulation in the leaf was positively and significantly correlated with rainfall during the curing season, especially between weeks 2 and 3 of curing which covers EOY and browning stages of curing with greatest nitrite and TSNA accumulation.;Rifampicin and ascorbic acid inhibited nitrosation in controlled curing when application was made at the beginning of the curing process. Chemical application at EOY tended to increase TSNA accumulation in the cured leaf. Ascorbic acid was the most effective blocker of TSNA accumulation in all treatment groups, but these results have not always been repeatable in conventional barn curing. |
