Tobacco-specific nitrosamines are carcinogenic N-nitrosamine compounds present at very low levels in freshly harvested tobacco leaves that accumulate during leaf curing. Formation of N-nitrosamine compounds is associated with high nitrate levels in the leaf at harvest, and nitrate is presumed to be the source from which the N-nitrosation species originates. More specifically, nitrite is considered to be a direct precursor, and nitrite is linked with N-nitrosation in many environmental matrices where it occurs via microbial nitrate reduction. Here, we initiate work exploring the role of leaf microbial communities in formation of tobacco-specific nitrosamines. Leaves from burley tobacco line TN90H were air cured under various temperature and relative humidity levels, and 22 cured tobacco samples were analyzed for their microbial communities and leaf chemistry. Analysis of nitrate, nitrite, and total tobacco-specific nitrosamine levels revealed a strong positive correlation between the three variables, as well as a strong positive correlation with increasing relative humidity during cure conditions. 16S rRNA gene amplicon sequencing was used to assess microbial communities in each of the samples. In most samples, Proteobacteria predominated at the phylum level, accounting for >90 % of the OTUs. However, a distinct shift was noted among members of the high tobacco-specific nitrosamine group, with increases in Firmicutes and Actinobacteria. Several OTUs were identified that correlate strongly (positive and negative) with tobacco-specific nitrosamine content. Copy number of bacterial nitrate reductase genes, obtained using quantitative PCR, did not correlate strongly with tobacco-specific nitrosamine content. Incomplete denitrification is potentially implicated in tobacco-specific nitrosamine levels.

Removal of terminal buds (topping) and control of the formation of axillary shoots (suckers) are common agronomic practices that significantly impact the yield and quality of various crop plants. Application of chemicals (suckercides) to plants following topping is an effective method for sucker control. However, our current knowledge of the influence of topping, and subsequent suckercide applications, to gene expression is limited. We analyzed the differential gene expression using RNA-sequencing in tobacco (Nicotiana tabacum) that are topped, or treated after topping by two different suckercides, the contact-localized-systemic, Flupro® (FP), and contact, Off-Shoot-T®. Among the differentially expressed genes (DEGs), 179 were identified as common to all three conditions. DEGs, largely related to wounding, phytohormone metabolism and secondary metabolite biosynthesis, exhibited significant upregulation following topping, and downregulation after suckercide treatments. DEGs related to photosynthetic processes were repressed following topping and suckercide treatments. Moreover, topping and FP-treatment affect the expression of auxin and cytokinin signaling pathway genes that are possibly involved in axillary shoot formation. Our results provide insights into the global change of plant gene expression in response to topping and suckercide treatments. The regulatory elements of topping-inducible genes are potentially useful for the development of a chemical-free sucker control system.

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Tobacco-specific nitrosamines (TSNAs) are known carcinogens in cured tobacco. They are produced primarily during the curing process, but agronomic practices occurring in the field as well as handling practices after curing may also influence TSNA levels, particularly if cured leaf is stored at high moisture. After curing and during market preparation, the cured leaf must be supple to avoid breakage. Ideally, this is after a period of wet weather during which the leaf absorbs moisture and comes into order or case. Often the weather remains dry for long periods after curing, and growers resort to artificial ordering to take down a sufficient amount of their crop to work on for several days, during which time the tobacco is bulked. The effect of this artificial ordering on TSNAs during short-term storage is not known. Field experiments were conducted in each of 3 years at two locations in Kentucky to evaluate TSNA accumulation following several ordering methods in dark air-cured and burley tobacco types. Treatments included natural ordering and variants of steaming and misting, which are both commonly used artificial ordering methods. At the Princeton location, samples were taken within 24 hr after the ordering treatments were done. In Lexington, samples were taken sequentially at takedown, after ordering, and after 14 d in the bulk. There were limited and inconsistent differences in total TSNAs between methods of ordering, and the TSNA levels were not affected by the moisture content of the leaf during bulking. There was a significant increase in TSNAs in the 24-hr period between takedown and bulking, which cannot be explained. We conclude that, in Kentucky, growers should use ordering methods that are best suited for their production system, but this may not be the case in warmer climates.