Blue mold, caused by the fungal pathogen Peronospora tabacina D.B. Adam, is one of the most important foliar diseases of tobacco (Nicotiana tabacum L.). Identification of molecular markers linked to genetic factors controlling resistance would facilitate development of resistant cultivars. Bulked segregant analysis was used to screen 1216 random amplified polymorphic DNA (RAPD) primers for their ability to reveal polymorphism between DNA bulks from susceptible doubled haploid (DH) lines and resistant DH lines possessing resistance derived from cultivar Ovens 62. Fifteen RAPD markers were tentatively identified as being linked to a major gene conditioning resistance to blue mold. These 15 markers (12 in coupling phase linkage with resistance and three in repulsion phase) were found to lie within a single linkage group of 36.6 cM and were subsequently tested on 122 DH lines derived from crosses between resistant and susceptible parents. F tests revealed statistically significant associations between resistance and each of the 15 RAPD markers. Interval mapping was used to more accurately place the quantitative trait locus (QTL) controlling resistance on the linkage map. The RAPD markers were screened on a set of 45 resistant and susceptible cultivars or breeding lines and four Nicotiana species. At variance with previous reports, marker genotypes indicated that resistance in Ovens 62 and most other blue mold resistant lines likely originated from N. debneyi Domin. Two RAPD markers flanking the most likely QTL position were converted to sequence characterized amplified region (SCAR) markers. These markers should aid in development of blue mold-resistant tobacco cultivars worldwide.

Bulked segregant (BSA) and random amplified polymorphic DNA (RAPD) analyses were used to identify markers linked to the dominant black shank resistance gene, Ph, from flue-cured tobacco (Nicotiana tabacum) cv. Coker 371-Gold. Sixty RAPD markers, 54 in coupling and 6 in repulsion phase linkage to Ph, were identified in a K 326-derived BC1F1 (K 326-BC1F1) doubled haploid (DH) population. Thirty RAPD markers, 26 in coupling and 4 in repulsion phase linkage to Ph, were used to screen 149 K 326-BC2F1 haploid plants. Complete linkage between the 26 coupling phase markers and Ph was confirmed by screening 149 K 326-BC2F1 DH lines produced from the haploid plants in black shank nurseries. RAPD markers OPZ-5770 in coupling and OPZ-7370 in repulsion phase linkage were used to select plants homozygous for the Ph gene for further backcrossing to the widely grown flue-cured cultivar K 326. Black shank disease nursery evaluation of 11 K 326-BC4S1 lines and their testcross hybrids to a susceptible tester confirmed linkage between Ph and OPZ-5770. The results demonstrated the efficiency of marker-assisted selection for Ph using a RAPD marker linked in coupling and repulsion. Complete linkage between 26 RAPD markers and the Ph gene was confirmed in the K 326-BC5 generation, and RAPD phenotypes were stable across generations and ploidy levels. These RAPD markers are useful in marker-assisted selection for Ph, an important black shank resistance gene in tobacco.

The successful introgression of transgenic virus resistance to control tomato spotted wilt virus (TSWV) epidemics relies on the stable inheritance of this type of resistance. We developed five segregating tobacco families transformed with sense or antisense copies of the TSWV nucleocapsid gene to determine the usefulness of TSWV-resistant transgenic parents in a breeding program, and to define the molecular determinants (transgene dosage, transcript and protein accumulation) controlling the expression of resistance. Three generations per family were evaluated: (1) R3 orR4 progeny derived from resistant transgenic parents used in crosses,(2) F1 hybrids from crosses between the resistant transgenic parents and untransformed tobacco, and (3) F2 progeny derived from resistantF1 plants. Analysis of two of these families revealed that resistance was correlated with either two insertions in hemizygous plants or a single homozygous insertion. In contrast, in two other families, there was no correlation between the number of insertions and the resistance response, but rather resistance was correlated with a single, actively transcribed insertion. Transgene protein accumulation was not detected in any of the plants analyzed, indicating that resistance is probably RNA-mediated. As inheritance of the transgene does not always result in the expression of resistance, special care may be necessary when using transgenic resistant parents in a breeding population.

Inheritance of resistance to the peanut root-knot nematode (Meloidogyne arenaria (Neal) Chitwood race 1) was investigated in the flue-cured tobacco cv. Speight G 28 and the breeding lines 81-RL-2K and SA 1214. The genetic relationship of this resistance in Speight G 28 to the resistance of the same cultivar to races 1 and 3 of M. incognita was also studied. Crosses were made between the root-knot nematode-susceptible flue-cured tobacco cv. NC 2326 and the three resistant genotypes. Parental, F1, F2 and backcross generations (BC1P1, BC1P2) were grown for each cross in randomized complete block designs with five replications in the greenhouse. Data indicated that resistance to M. arenaria race 1 in the three resistance sources is conditioned by a single dominant gene, but this resistance is partial compared to that for M. incognita races 1 and 3. Further, resistance to races 1 and 3 of M. incognita and resistance to M. arenaria race 1 in cv. Speight G 28 appear to be controlled by the same gene. These results, combined with the absence of segregation in the F2 populations of the crosses between resistant parents 81-RL-2K × SA 1214, 81-RL-2K × Speight G 28, and SA 1214 × Speight G 28, suggest allelism of resistance among these genotypes.

Resistance to the southern root-knot nematode, Meloidogyne incognita races 1 and 3, has been identified, incorporated, and deployed into commercial cultivars of tobacco, Nicotiana tabacum. Cultivars with resistance to other economically important root-knot nematode species attacking tobacco, M. arenaria, M. hapla, M. javanica, and other host-specific races of M. incognita, are not available in the United States. Twenty-eight tobacco genotypes of diverse origin and two standard cultivars, NC 2326 (susceptible) and Speight G 28 (resistant to M. incognita races 1 and 3), were screened for resistance to eight root-knot nematode populations of North Carolina origin. Based on root gall indices at 8 to 12 weeks after inoculation, all genotypes except NC 2326 and Okinawa were resistant to M. arenaria race 1, and races 1 and 3 of M. incognita. Except for slight root galling, genotypes resistant to M. arenaria race 1 responded similarly to races 1 and 3 of M. incognita. All genotypes except NC 2326, Okinawa, and Speight G 28 showed resistance to M. javanica. Okinawa, while supporting lower reproduction of M. javanica than NC 2326, was rated as moderately susceptible. Tobacco breeding lines 81-R-617A, 81-RL- 2K, SA 1213, SA 1214, SA 1223, and SA 1224 were resistant to M. arenaria race 2, and thus may be used as sources of resistance to this pathogen. No resistance to M. hapla and only moderate resistance to races 2 and 4 of M. incognita were found in any of the tobacco genotypes. Under natural field infestations of M. arenaria race 2, nematode development on resistant tobacco breeding lines 81-RL-2K, SA 1214, and SA 1215 was similar to a susceptible cultivar with some nematicide treatments; however, quantity and quality of yield were inferior compared to K 326 plus nematicides.

Random amplified polymorphic DNA (RAPD) analysis was conducted to map the Rk gene in tobacco which conditions resistance to races 1 and 3 of the root-knot nematode, Meloidogyne incognita. Resistant burley tobacco genotype NC 528, containing the Rk gene, and the susceptible cultivar Ky 14 were screened with 1,500 random decamers. A low rate of genetic polymor-phism (<10%) was detected among these lines. Two populations (F1 and F3) of maternally de-rived doubled haploid (MDH) lines of burley tobacco, developed from the cross NC 528 × Ky 14, were used to map the Rk gene. NC 528, Ky 14, three Rk-resistant (Rk-R) DNA bulks, andthree Rk-susceptible (Rk-S) bulks generated from F1-derived MDH individuals were screenedwith the primers that amplified bands polymorphic between Rk-R and Rk-S lines. A total of 67 F1MDH lines and 59 F3MDH lines were screened with the primers that amplified bands polymorphic between Rk-R bulks and Rk-S bulks to confirm linkage between candidate markers and the Rk gene. Sixteen RAPD markers were positioned at six loci in a map 24.1 centimorgans long. Six RAPD markers, including one identified in the F3MDH population, were mapped at the Rk locus.

Cultivars with moderate (KY 14), low (Burley 21), and no resistance (Judy's pride) to black root rot, and the F-1 of gy 14 x Burley 21 were evaluated for components of partial resistance to Thielaviopsis basicola. In addition, transgressive segregants from a previous generation mean analysis were intercrossed and progeny of the following crosses were evaluated: (KY 14 x Burley 21)F-2 x (KY 14 x (KY 14 x Burley 21)F-1), (KY 14 x Burley 21)F-2 x (Burley 21 x (KY 14 x Burley 21)F-1), and (KY 14 x (KY 14 x Burley 21)F-1) x (Burley 21 x (KY 14 x Burley 21)F-1). Five-week-old seedlings were transplanted into soil infested with 100 chlamydospores of T. basicola per gram of soil mixture and grown in the greenhouse at an average air temperature of 21 degrees C. Disease severity (percent root necrosis), number of lesions per root, lesion length, and population density of the pathogen were estimated 3 weeks after transplanting. Significant differences were observed among genotypes for each component of partial resistance measured. Significant positive correlations were observed between lesion number and disease severity and between lesion number and lesion length. However, a significant negative correlation was observed between lesion number and population density. Selection for components of partial resistance in burley tobacco should lead to increased levels of resistance to black root rot.

Ng'ambi, T. B. S., Rufty, R. C., and Barker, K. R. 1995. Response of root-knot resistant tobacco to concomitant populations of Meloidogyne species. Plant Dis. 79:1008-1013. Mixed populations of Meloidogyne species commonly occur in most flue-cured tobacco (Nicotiana tabacum L.) fields in the southeastern United States. The interaction of Meloidogyne arenaria race 2 (Ma) and M. incognita race 3 (Mi) on the Mi-resistant flue-cured tobacco cv. Speight G-28 and on a root-knot susceptible flue-cured tobacco cv. NC 2326 (control) was evaluated under greenhouse conditions. Plants of both cultivars were inoculated with 1,500 or 3,000 eggs of Ma or Mi alone, or with a combination of 1,500 eggs of each species. Root penetration (number of juveniles per g of root) with the mixed population assessed 10 days after inoculation (DAI) was comparable to that of Ma alone on the Mi-resistant tobacco cultivar, and of either nematode species alone on the susceptible cultivar. Also, nematode population density (number of nematodes per g of root, all stages except eggs), numbers of mature females per g of root, root damage (percent root area galled), and nematode reproduction (number of eggs per g of root) on Speight G-28 30 and/or 48 DAI with mixed population were not greater than those attributable to Ma alone. Indeed, inoculation with the mixed population resulted in slightly lower nematode population density (except at 48 DAI), root damage, and nematode reproduction on Mi-resistant cultivar than with Ma alone at the 1,500 egg inoculum level. The concomitant inoculation of Ma and Mi on NC 2326 did not differ from single inoculation with Ma for all variables measured, except reproduction at 48 DAI. These data indicate that Speight G-28 resistance to Mi was not reduced with concomitant infections of Ma and Mi. Fewer Mi than Ma mature females were recovered from the susceptible cultivar in the mixed population, indicating that Ma was more competitive than Mi.

Tobacco etch virus (TEV) and tobacco vein mottling virus (TVMV) are serious diseases of burley tobacco and are controlled most effectively by use of resistant cultivars. The inheritance of resistance to TEV and TVMV in the burley 'tobacco cultivar Sota 6505 and allelism with other sources of potyvirus resistance were evaluated. Crosses were made between Sota 6505 and two susceptible burley tobacco cultivars, Ky 14 and Va 528. Parental genotypes, F1, F2, and backcross generations to each of the parents were evaluated in randomized complete block designs for TEV resistance at Waynesville, North Carolina, and Las Varas, Nayarit, Mexico, and for TVMV resistance at Laurel Springs, North Carolina, during 1988 and 1989. Crosses were also made between Sota 6505 and TEV- and TVMV-resistant cultivars Virgin A Mutant and Havana 307 to test for allelism. Resistant X resistant crosses were evaluated for TEV resistance in a greenhouse in Raleigh, North Carolina. Plants were mechanically inoculated in the field approximately 1 mo after transplanting and in the greenhouse 1 wk after transplanting. Disease severity data were collected at topping time in the field and 1 mo after transplanting in the greenhouse. Chi-square goodness of fit tests were conducted, but simple Mendelian inheritance ratios did not fit the data for expression of resistance with either virus, possibly because of environmental effects. Generation means analysis showed that a simple additive-dominance model adequately described the data. Additivity was the major genetic effect, and there was no evidence of epistasis. The gene(s) controlling TEV resistance in Sota 6505 appears to be allelic to the virus resistance gene(s) found in resistant cultivars Virgin A Mutant and Havana 307.

Anther-derived doubled haploid (ADH) tobacco lines possessing a high level of resistance to tobacco black shank, Phytophthora parasitica (Dast.) var ‘nicotianae’ (B. de Haan) Tucker (Ppn), have been identified from a cross of two tobacco cultivars susceptible to this disease. The objective of this study was to investigate the origin of black shank resistance in ADH lines developed from two susceptible parental cultivars: ‘Ovens 62’ and ‘Ky 15’. In addition to the ADH lines, sexually-derived F2∶8 lines were produced using the single seed descent (SSD) method. Seventy-five ADH lines and 75 SSD lines along with two black shank resistant and three susceptible controls (including parental cultivars) were evaluated under field conditions for resistance to Ppn in 1989 and 1990. Lines were assigned at random to five sets and were planted in a randomized complete block design with three replications/set. A disease index was computed from weekly stand counts of the number of surviving plants per plot for each tobacco line. The 1989 experiment revealed resistance to Ppn in 8 ADH lines, numbers 29, 31, 32, 40, 68, 69, 74, 76, and 1 SSD line, number 32. The 8 ADH lines continued to show resistance in 1990, but SSD line number 32 exhibited a susceptible reaction. There were no other resistant SSD lines.

Potato virus Y (PVY), susceptible tobacco (Nicotiana tabacum L.) cultivar, McNair 944, was subjected to in vitro anther culture to determine if genetic variability for virus resistance could be induced among resulting haploids. Five hundred and forty-five haploids were produced and inoculated with a highly necrotic strain (NN) of PVY. One haploid plant survived, even though it was infected with the virus. Selfed progenies of a chromosome-doubled plant of this variant, designated NC 602, proved to be highly resistant to the necrotic effects of the virus. An investigation into the genetic nature of this variant showed the resistance mechanism to be controlled by a single gene exhibiting incomplete dominance. Cytoplasmic and maternal effects were not involved in the disease resistance reaction. The variant was challenged with ten additional strains of PVY from an international collection, and it proved to be resistant to three (VAM-B, MM, and Spanish) strains. NC 602 was evaluated for five agronomic traits and concentrations of total alkaloids as nicotine and reducing sugars in cured leaf. The gametoclonal variant differed from McNair 944 only for cured leaf yield, where an 18.4% reduction was measured.

Crop ScienceVolume 30, Issue 1 cropsci1990.0011183X003000010071x p. 241-242 Registration of Germplasms Registration of NC-BMR 42 and NC-BMR 90 Germplasm Lines of Tobacco R. C. Rufty, Corresponding Author R. C. Rufty n/[email protected] Dep. of Crop ScienceCorresponding author.Search for more papers by this authorE. A. Wernsman, E. A. Wernsman Dep. of Crop ScienceSearch for more papers by this authorC. E. Main, C. E. Main Dep. of Crop ScienceSearch for more papers by this authorG. V. Gooding Jr., G. V. Gooding Jr. Dep. of Plant Pathology, North Carolina State University, Raleigh, NC, 27695Search for more papers by this author R. C. Rufty, Corresponding Author R. C. Rufty n/[email protected] Dep. of Crop ScienceCorresponding author.Search for more papers by this authorE. A. Wernsman, E. A. Wernsman Dep. of Crop ScienceSearch for more papers by this authorC. E. Main, C. E. Main Dep. of Crop ScienceSearch for more papers by this authorG. V. Gooding Jr., G. V. Gooding Jr. Dep. of Plant Pathology, North Carolina State University, Raleigh, NC, 27695Search for more papers by this author First published: 01 January 1990 https://doi.org/10.2135/cropsci1990.0011183X003000010071xCitations: 5AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL No abstract is available for this article.Citing Literature Volume30, Issue1January–February 1990Pages 241-242 RelatedInformation

A 5-parent full diallel and a 9-parent half diallel including adapted and exotic wheats of both winter and spring types were evaluated in the greenhouse for components of partial resistance to S. [Leptosphaeria] nodorum. F1 seedlings and parents were inoculated with a mixture of isolates of L. nodorum and evaluated for inheritance of incubation period and infection frequency. Significant general combining ability effects were found for incubation period and infection frequency. Significant specific combining ability effects were found for incubation period, but were observed in only 40% of the hybrid combinations in the full diallel and 16% of the hybrid combinations in the half diallel. Additive gene effects were most important in the inheritance of both components. Reciprocal and maternal effects were non-significant for both components of partial resistance measured by increasing incubation period and decreasing infection frequency. Another partially resistant parent (PAT 72-180) contributed to resistance through an increased incubation period only

A suspension fo Pseudomonas syringae pv. tabaci containing 10 7 cfu/ml produced symptoms typical of those observed in the field. A preinoculation mist period of 0-18 hr did not increase the amount of disease. Postinoculation mist periods of 24,36, and 48 hr signigicantly incrases the amount of disease assessment scale was developed and used successfully to classify genotypes for disease resistance

Rufty, R. C., and Main, C. E. 1989. Components of partial resistance to blue mold in six tobacco genotypes under controlled environmental conditions. Phytopathology 79:606-609. Two susceptible and four partially resistant tobacco genotypes were Mutant, NC-BMR 42, and NC-BMR 90 produced fewer and smaller evaluated for components of partial resistance to blue mold. Disease lesions, had lower sporulation capacity, and exhibited longer patent efficiency, incubation period, latent period, degree of colonization, and periods than susceptible genotypes. Breeding line NC-BMR 90 was sporulation capacity were measured under high and low inoculation superior to all other partially resistant genotypes in this study. The presence density in experiments conducted at the Southeastern Plant Environmental of multiple resistance components in NC-BMR 90 makes this line a Laboratory (North Carolina State University Phytotron), Raleigh, NC. desirable source of blue mold resistance. Results obtained in these Genotypes differed significantly for all components of resistance in all experiments corroborated field observations and indicate that deployment trials. Commercial cultivars Speight G-70 and McNair 944 were of tobacco germ plasm with partial resistance to blue mold should reduce consistently the most susceptible genotypes based on all components of onset and progress of blue mold epidemics. partial resistance measured. Partially resistant genotypes Chemical Additional keywords: Nicotiana tabacum, Peronospora tabacina. Tobacco blue mold, also known as downy mildew of tobacco derived from N. velutina) and Chemical Mutant (resistance (Nicotiana tabacum L.), is caused by the fungus Peronospora obtained after treating seeds of flue-cured tobacco cultivar tabacina Adam. Occurrence of blue mold is generally sporadic, but Virginia Gold with triethylene iminotriazine) (9); two resistant severe economic losses can occur when the disease reaches breeding lines, NC-BMR 42 (derived from the cross Ovens 62 X epidemic proportions. For example, an epidemic of blue mold in McNair 944) and NC-BMR 90 (derived from the cross Ovens 62 X 1979 resulted in an estimated $250 million loss to U.S. and KY 17) (12); and two susceptible commercial cultivars, Speight

Cultivar development by doubled haploid (DH) methods employing in vitro anther culture has not been highly successful in tobacco, Nicotiana tabacum L., because of the low yielding ability of DH lines. The objective of this study was to compare DH lines developed from androgenetic and gynogenetic haploid sporophytes from the same parental plants. Single plants from ‘Ky10’, ‘Ky15’, and ‘Kyl7’ were self‐pollinated, crossed with pollen of N. africana Merx. and Buttler, and immature buds were anther cultured. Haploid sporophytes arising from embryo sac nuclei and vegetative nuclei of pollen were chromosome doubled and self‐pollinated. Ten random androgenetic DH lines (ADHs), 10 random gynogenetic DH lines (MDHs), and selfed progenies of the parental plant were assigned to a set, or cultivar family, and grown in three replications in each set in two environments. Data were collected for eight characters. Sixteen second cycle MDH lines were produced from one random first cycle line, Ky 10 MDH 1, and compared with the original Ky 10 parent, a first cycle MDH line composite, and Ky 10 MDH 1. The ADH and MDH lines were significantly different for all characters measured. The MDH lines were agronomically superior to ADH lines and more closely resembled the cultivars. The ADHs and MDHs yielded 79.9% and 91.5%, respectively, of the cultivars. A second cycle of MDH line production resulted in no further yield reduction. A model of heterozygosity and inbreeding depression in the parental cultivars could explain the results for yield of the MDH lines; it would not account for differences between ADH and MDH lines.

Burley tobacco is susceptible to several different types of virus diseases that suppress plant growth and development. Two viruses, tobacco etch virus (TEV) and tobacco vein mottling virus (TVMV), are particularly damaging to burley. Burley tobacco cultivars resistant to these two viruses are currently being developed. Some of these cultivars also show differential sensitivity to ozone (O3). Recent field observations have suggested that burley tobacco infected with TEV and TVMV was more sensitive to O3 than non-virus-infected tobacco. Experiments were designed to identify interactions between O3 and each of the two virus diseases. Three cultivars, Burley 21, Burley 49, and Greeneville 131, which were differentially sensitive to O3 and both virus diseases, were grown in a charcoal-filtered greenhouse environment. Tobacco plants of each cultivar were inoculated with TEV or TVMV, and virus infected and virus-free plants were exposed to 0·0, 0·05, 0·2, and 0·4 ppm O3 (1 ppm of O3 is equivalent to 1960 μg m−3), 3h day−1, 5 days week−1 for 3 weeks in continuous-stirred tank reactor exposure chambers in the greenhouse. Exposures were begun after systemic virus symptoms were expressed in inoculated plants. The suppression of lead and stem dry weight by increasing O3 concentrations was less in TEV-infected burley cultivars than in noninfected burley cultivars. Tobacco vein mottling virus infection enhanced biomass suppression by O3 on Burley 21 and on Greeneville 131, but not on Burley 49. Thus, the interactions with O3 were dependent on specific virus-cultivar combinations.