Genetic and Agronomic Analysis of Tobacco Genotypes Exhibiting Reduced Nicotine Accumulation Due to Induced Mutations in Berberine Bridge Like (BBL) Genes.

Genetic methodologies for reducing nicotine accumulation in the tobacco plant (Nicotiana tabacum L.) are of interest because of potential future regulations that could mandate lowering of this alkaloid in conventional cigarettes. Inactivation of tobacco genes such as the Berberine Bridge Like (BBL) gene family believed to encode for enzymes involved in one of the latter steps of nicotine biosynthesis could be a viable strategy for producing new tobacco cultivars with ultra-low leaf nicotine accumulation. We introduced deleterious mutations generated via ethyl methanesulfonate treatment of seed or gene editing into six known members of the BBL gene family and assembled them in different combinations to assess their relative contribution to nicotine accumulation. Significant reductions (up to 17-fold) in percent leaf nicotine were observed in genotypes homozygous for combined mutations in BBL-a, BBL-b, and BBL-c. The addition of mutations in BBL-d1, BBL-d2, and BBL-e had no additional significant effect on lowering of nicotine levels in the genetic background studied. Reduced nicotine levels were associated with reductions in cured leaf yields (up to 29%) and cured leaf quality (up to 15%), evidence of physiological complexities within the tobacco plant related to the nicotine biosynthetic pathway. Further nicotine reductions were observed for a BBL mutant line cultivated under a modified production regime in which apical inflorescences were not removed, but at the expense of further yield reductions. Plants in which BBL mutations were combined with naturally occurring recessive alleles at the Nic1 and Nic2 loci exhibited further reductions in percent nicotine, but no plant produced immeasurable levels of this alkaloid. Findings may suggest the existence of a minor, alternative pathway for nicotine biosynthesis in N. tabacum. The described genetic materials may be of value for the manufacture of cigarettes with reduced nicotine levels and for future studies to better understand the molecular biology of alkaloid accumulation in tobacco.

Characterization of Phytophthora nicotianae Resistance Conferred by the Introgressed Nicotiana rustica Region, Wz, in Flue-Cured Tobacco.

Black shank, caused by Phytophthora nicotianae, is one of the most important diseases affecting tobacco worldwide and is primarily managed through use of host resistance. An additional source of resistance to P. nicotianae, designated as Wz, has been introgressed into Nicotiana tabacum from N. rustica. The Wz gene region confers high levels of resistance to all races, but has not been characterized. Our study found Wz-mediated resistance is most highly expressed in the roots, with only a slight reduction in stem-lesion size in Wz genotypes compared with susceptible controls. No substantial relationships were observed between initial inoculum levels and disease development on Wz genotypes, which is generally consistent with qualitative or complete resistance. Isolates of P. nicotianae adapted for five host generations on plants with the Wz gene caused higher disease severity than isolates adapted on Wz plants for only one host generation. Wz-adapted isolates did not exhibit increased aggressiveness on genotypes with other sources of partial resistance, suggesting pathogen adaptation was specific to the Wz gene. To reduce potential for pathogen population shifts with virulence on Wz genotypes, Wz should be combined with other resistance sources and rotation of varying black shank resistance mechanisms is also recommended.

Multiple Disease Resistance Loci Affect Soilborne Disease Resistance in Tobacco (Nicotiana tabacum).

Phytophthora nicotianae and Ralstonia solanacearum are two of the most important pathogens affecting tobacco worldwide. Greater insight regarding genetic systems controlling resistance to these two soilborne pathogens, as well as identification of DNA markers associated with genomic regions controlling this resistance, could aid in variety development. An evaluation of 50 historical tobacco lines revealed a high positive correlation between resistances to the two pathogens, preliminarily suggesting that some genomic regions may confer resistance to both pathogens. A quantitative trait loci (QTL) mapping experiment designed to investigate the genetic control of soilborne disease resistance of highly resistant 'K346' tobacco identified four QTL significantly associated with resistance to P. nicotianae (explaining 60.0% of the observed phenotypic variation) and three QTL to be associated with R. solanacearum resistance (explaining 50.3% of the observed variation). The two QTL with the largest effect on Phytophthora resistance were also found to be the QTL with the greatest effects on resistance to Ralstonia. This finding partially explains previously observed associations between resistances to these two pathogens among U.S. current cultivars and within breeding populations. Further study is needed to determine whether these relationships are due to the same genes (i.e., pleiotropy) or favorable coupling-phase linkages that have been established over time.

The Tobacco Trichome Exudate Z-abienol and Its Relationship With Plant Resistance to Phytophthora nicotianae.

In previous research, we discovered a favorable quantitative trait locus (QTL) in cigar tobacco cultivar 'Beinhart 1000' designated as Phn15.1, which provides a high level of partial resistance to the black shank disease caused by Phytophthora nicotianae. A very close genetic association was also found between Phn15.1 and the ability to biosynthesize Z-abienol, a labdanoid diterpene exuded by the trichomes onto above-ground plant parts, and that imparts flavor and aroma characteristics to Oriental and some cigar tobacco types. Because accumulation of Z-abienol is considered to be undesirable for cultivars of other tobacco types, we herein describe a series of experiments to gain insight on whether this close association is due to genetic linkage or pleiotropy. First, in an in vitro bioassay, we observed Z-abienol and related diterpenes to inhibit hyphal growth of P. nicotianae at concentrations between 0.01 and 100 ppm. Secondly, we field-tested transgenic versions of Beinhart 1000 carrying RNAi constructs for downregulating NtCPS2 or NtABS, two genes involved in the biosynthesis of Z-abienol. Thirdly, we also field tested a recombinant inbred line population segregating for a truncation mutation in NtCPS2 leading to an interrupted Z-abienol pathway. We observed no correlation between field resistance to P. nicotianae and the ability to accumulate Z-abienol in either the transgenic materials or the mapping population. Results suggest that, although Z-abienol may affect P. nicotianae when applied at high concentrations in in vitro assays, the compound has little effect on black shank disease development under natural field conditions. Thus, it should be possible to disassociate Phn15.1-mediated black shank resistance identified in cigar tobacco cultivar Beinhart 1000 from the ability to accumulate Z-abienol, an undesirable secondary metabolite for burley and flue-cured tobacco cultivars.