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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 BC 1 F 1 (K 326-BC 1 F 1 ) 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-BC 2 F 1 haploid plants. Complete linkage between the 26 coupling phase markers and Ph was confirmed by screening 149 K 326-BC 2 F 1 DH lines produced from the haploid plants in black shank nurseries. RAPD markers OPZ-5 770 in coupling and OPZ-7 370 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-BC 4 S 1 lines and their testcross hybrids to a susceptible tester confirmed linkage between Ph and OPZ-5 770 . 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-BC 5 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.

ABSTRACT Transformation of plants with the nucleocapsid (N) gene of Tomato spotted wilt tospovirus (TSWV) provides resistance to disease development; however, information is lacking on the response of plants to natural inoculum in the field. Three tobacco cultivars were transformed with the N gene of a dahlia isolate of TSWV (TSWV-D), and plants were evaluated over several generations in the greenhouse. The resistant phenotype was more frequently observed in 'Burley 21' than in 'KY-14' or 'K-326', but highly resistant 'Burley 21' transgenic lines were resistant to only 44% of the heterologous TSWV isolates tested. Advanced generation (R(3) and R(4)) transgenic resistant lines of 'Burley 21' and a 'K-326' F(1) hybrid containing the N genes of two TSWV isolates were evaluated in the field near Tifton, GA, where TSWV is endemic. Disease development was monitored by symptom expression and enzyme-linked immunosorbent assay (ELISA) analysis. Whereas incidence of TSWV infection in 'Burley 21' susceptible controls was 20% in 1996 and 62% in 1997, the mean incidence in transgenic lines was reduced to 4 and 31%, respectively. Three transgenic 'Burley 21' lines were identified that had significantly lower incidence of disease than susceptible controls over the two years of the study. In addition, the rate of disease increase at the onset of the 1997 epidemic was reduced for all the 'Burley 21' transgenic lines compared with the susceptible controls. The 'K-326' F(1) hybrid was as susceptible as the 'K-326' nontransformed control. ELISA analysis demonstrated that symptomless plants from the most resistant 'Burley 21' transgenic lines accumulated detectable nucleocapsid protein, whereas symptomless plants from more susceptible lines did not. We conclude that transgenic resistance to TSWV is effective in reducing incidence of the disease in the field, and that accumulation of transgene protein may be important in broad-spectrum resistance.

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.

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, F 1 , F 2 and backcross generations (BC 1 P 1 , BC 1 P 2 ) 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 F 2 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.

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 (F 1 and F 3 ) 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 F 1 -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.

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.A second field experiment was conducted in 1990 using the putative resistant ADH lines and SSD line nr. 32, which expressed resistance to black shank in 1989. In addition, 12 randomly selected lines from the original 150 ADH and SSD lines were evaluated along with the same five controls as in the previous experiment to further substantiate the resistance of the ADH lines. Lines were planted in a randomized complete block design with eight replications. ADH lines continued to express resistance. SSD line nr. 32 did not show any resistance nor did any other line derived through the single seed descent procedure.These results support the hypothesis that anther culture can generate a high level of black shank resistance. The basis for this resistance is postulated to be due to gene amplification of factors that regulate host response to the black shank fungus.

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.

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 microg 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.