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Published online 1 September 2007
Published in Crop Sci 47:1975-1984 (2007)
© 2007 Crop Science Society of America
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PLANT GENETIC RESOURCES

Evaluation of Nicotiana tabacum Genotypes Possessing Nicotiana africana-derived Genetic Tolerance to Potato Virus Y

R. S. Lewis*

Campus Box 7620, Dep. of Crop Science, North Carolina State Univ., Raleigh, NC 27695

* Corresponding author (ramsey_lewis{at}ncsu.edu).


   ABSTRACT
TOP
 NOTES
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
New alleles influencing resistance to potato virus Y (PVY) would be valuable for developing resistant tobacco (Nicotiana tabacum L.) cultivars. The first objective of this research was to evaluate materials possessing an introgressed genomic region (Nafr) from N. africana Merx. & Buttler for their resistance against an array of nine PVY isolates. Seven near-isogenic genotypes of flue-cured tobacco cultivar K326 were produced that possessed Nafr and the recessive resistance gene, va, in different combinations and zygosities. Nafr provided little protection against mild non-necrotic isolates, but imparted significant increased resistance against the necrotic effects of two severe isolates. Data indicated that Nafr and va can be combined to increase the range and level of resistance or tolerance to PVY. A second objective was to evaluate five backcross-derived K326 Nafr/Nafr isolines and corresponding hemizygous F1 hybrids for yield and quality characteristics in field experiments in the absence of PVY infection. The K326 Nafr/— genotypes were not significantly different from K326 for yield or cash return, and produced cured leaf with improved quality. The Nafr/Nafr genotypes were inferior to Nafr/— genotypes for yield, cash return, and cured leaf quality. Genotypes of the Nafr/— va/va combination may have the greatest practical value when yield, quality, and reaction to PVY infection are collectively taken into consideration.

Abbreviations: PVY, potato virus Y • RAPD, random amplified polymorphic DNA

Evaluation of Nicotiana tabacum Genotypes Possessing Nicotiana africana-derived Genetic Tolerance to Potato Virus Y

R. S. Lewis*

Campus Box 7620, Dep. of Crop Science, North Carolina State Univ., Raleigh, NC 27695

* Corresponding author (ramsey_lewis{at}ncsu.edu).

New alleles influencing resistance to potato virus Y (PVY) would be valuable for developing resistant tobacco (Nicotiana tabacum L.) cultivars. The first objective of this research was to evaluate materials possessing an introgressed genomic region (Nafr) from N. africana Merx. & Buttler for their resistance against an array of nine PVY isolates. Seven near-isogenic genotypes of flue-cured tobacco cultivar K326 were produced that possessed Nafr and the recessive resistance gene, va, in different combinations and zygosities. Nafr provided little protection against mild non-necrotic isolates, but imparted significant increased resistance against the necrotic effects of two severe isolates. Data indicated that Nafr and va can be combined to increase the range and level of resistance or tolerance to PVY. A second objective was to evaluate five backcross-derived K326 Nafr/Nafr isolines and corresponding hemizygous F1 hybrids for yield and quality characteristics in field experiments in the absence of PVY infection. The K326 Nafr/— genotypes were not significantly different from K326 for yield or cash return, and produced cured leaf with improved quality. The Nafr/Nafr genotypes were inferior to Nafr/— genotypes for yield, cash return, and cured leaf quality. Genotypes of the Nafr/— va/va combination may have the greatest practical value when yield, quality, and reaction to PVY infection are collectively taken into consideration.

Abbreviations: PVY, potato virus Y • RAPD, random amplified polymorphic DNA


   INTRODUCTION
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
POTATO VIRUS Y (PVY) belongs to the largest plant virus family, Potyviridae, and is one of the most economically important pathogens affecting tobacco (Nicotiana tabacum L.) production worldwide (Lucas, 1975; Delon et al., 1993). Genetic resistance offers the most efficient means of reducing economic loss from crop plant viruses. Extremely high levels of resistance to many viruses can be generated in a number of crop species, including tobacco, using transgenic expression of virus-derived nucleotide sequences (Lawson et al., 1990; Sudarsono et al., 1995; Dinant et al., 1998). Current international objection to genetically engineered tobacco cultivars makes commercialization of these technologies difficult, however. Resistance to many PVY isolates (as well as other potyviruses such as tobacco etch virus and tobacco vein mottling virus) is conferred by irradiation-induced recessive alleles at the va locus in ‘Virgin A Mutant’ (Koelle, 1961), and also by naturally occurring recessive alleles at this locus found in several other cultivars (Wernsman, 1992a). Partial resistance has also been identified in gametoclonal variant NC602 (Witherspoon et al., 1991). No source provides complete resistance to all strains or isolates of PVY, however. New sources of genetic variability influencing resistance or tolerance to this important pathogen are therefore continually being sought.

Lucas et al. (1980) found an accession of the African Nicotiana species N. africana to be immune to three strains of PVY. Through interspecific hybridization and backcrossing, an alien chromosome segment acting in a partially dominant fashion to confer tolerance to a severe isolate of PVY was transferred from this species to N. tabacum (Wernsman, 1992b; Lewis, 2005). Materials possessing this region are considered tolerant to this isolate because they resist systemic necrosis that occurs in PVY-susceptible genotypes. A set of random amplified polymorphic DNA (RAPD) markers contained within the introgressed region were also identified (Lewis, 2005).

Potato virus Y is a highly variable pathogen, and numerous strains elicit a wide array of reactions across various tobacco genotypes. The effectiveness of the introgressed N. africana genomic region against diverse PVY isolates has not yet been reported. The first objective of this research was to investigate the effectiveness of the alien chromosome segment against a set of nine PVY isolates collected from diverse tobacco-growing regions of the world, and that differ widely in terms of symptom type and severity on tobacco. Evaluations were based on symptom development in environmentally controlled growth chamber experiments. The alien segment was evaluated in hemizygous and homozygous conditions, and also in combination with the recessive gene, va, derived from tobacco breeding line ‘Greenville 136.’ Since modifying genes can influence observed reactions to virus infections (Johnson et al., 1982; Reddick et al., 1991), these experiments were performed using nearly isogenic materials possessing the genetic mechanisms in different combinations in a ‘K326’ genetic background.

Tobacco breeding lines possessing disease resistance genes transferred from wild relatives have often had reduced commercial value because of reduced yields or quality associated with the introgressed chromatin. This is probably due to linkage drag effects caused by deleterious genes of alien origin flanking the resistance gene (Chaplin et al., 1966; Legg et al., 1981; Johnson, 1999; Lewis et al., 2007). Deployment of such genes is often done in hemizygous condition to lessen unfavorable effects (Wernsman and Rufty, 1987). A second objective of this research was to conduct field evaluations of genetic materials possessing the introgressed N. africana region in both homozygous and hemizygous condition to determine if its presence was associated with reduced yields or cured leaf quality.


   MATERIALS AND METHODS
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Evaluation of Resistance in Growth Chamber Experiments
Plant Material
To allow comparison of different resistance or tolerance gene combinations in a nearly isogenic background, both va and the introgressed N. africana-derived genomic region (hereafter referred to as Nafr) were transferred to the popular, high-yielding PVY-susceptible flue-cured tobacco cultivar K326 using backcrossing. The K326 va/va genotype was developed by transferring the recessive va allele contained in burley tobacco breeding line Greenville 136 to K326 using eight backcrosses (with test crossing at each stage of backcrossing to confirm the presence of va), followed by self-pollination to establish a homozygous va/va isoline.

A 2n = 48 plant homozygous for Nafr was previously developed by Lewis (2005). The alien segment was transferred to K326 using five backcrosses with selection for resistance against the necrotic effects of an isolate of PVY strain NSNR (designated as NC78) maintained by the tobacco breeding program at North Carolina State University (Gooding and Tolin, 1973). The Nafr region is transmitted through male gametes at a low frequency (Lewis, 2005). To achieve homozygosity for the region, an array of maternally derived haploid plants was generated from a PVY NSNR–tolerant BC5F1 individual using the method of Burk et al. (1979a). Haploid plants possessing Nafr were identified based on the presence of two N. africana RAPD marker loci, UBC119.927 and OPZ20.342 (Lewis, 2005). Recombination between PVY NSNR tolerance and these markers has not been observed (Lewis, 2005). Selected individuals were chromosome doubled using the method of Kasperbauer and Collins (1972). Potato virus Y tolerance of the resulting doubled haploid lines was verified via inoculation with PVY isolate NC78. One line, designated as DH04B-702–1, was selected to represent K326 Nafr/Nafr for the growth chamber experiments (Table 1 ).


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  		Table 1. Genotypes used in experiments to evaluate cv. K326 and derived isolines for reactions to potato virus Y inoculation.

 
To combine va with Nafr, DH04B-702–1 was hybridized with K326 va/va. An array of maternally derived haploid plants was generated from this cross, and individuals carrying Nafr were selected based on genotypes at the UBC119.927 and OPZ20.342 RAPD marker loci. These individuals were chromosome doubled, and derived doubled haploid lines were inoculated with PVY NSNR (isolate NC78) to identify Nafr/Nafr lines that were also homozygous for va. Lines carrying only Nafr are resistant to the necrotic effects of this isolate, but exhibit obvious leaf mottling within 10 d after inoculation. The Nafr/Nafr va/va genotypes exhibit an obviously superior level of resistance to the NC78 isolate of PVY NSNR, and typically exhibit no symptoms until about 16 d, when slight breakdown of the va/va mechanism occurs. Doubled haploid line DH05B-515–15 was selected to represent the K326 Nafr/Nafr va/va genotype for the growth chamber experiments (Table 1).

Various crosses between K326 and the three nearly isogenic lines described above were used to produce four F1 hybrids carrying va and Nafr in different combinations and zygosities (Table 1). Cultivar K326 was hybridized with K326 va/va to create an F1 hybrid designated as K326 Va/va. Doubled haploid line DH04B-702–1 was crossed with K326 to produce an F1 hybrid hemizygous for Nafr (K326 Nafr/—). Doubled haploid line DH05B-515–15 was hybridized with K326 va/va to produce a K326 Nafr/— va/va F1 hybrid. Finally, DH05B-515–15 was crossed with K326 to produce a K326 Nafr/— Va/va F1 hybrid.

Virus Isolates and Inoculations
Nine PVY isolates were selected to represent diversity in origin, symptom development on N. tabacum (ranging from mild mottling to systemic necrosis), and reaction on va/va genotypes (some va-breaking strains were selected). These isolates and their descriptions are listed in Table 2 . Virus isolates were maintained in tobacco cultivar Burley 21 in insect-proof cages in a greenhouse, and inoculum was prepared by macerating systemically infected leaf in phosphate buffer (0.05 mol L–1 Na2HPO4–KH2PO4, pH 7.2, 1 g tissue:5 mL buffer) using a mortar and pestle. Approximately 1% (w/v) carborundum (600 mesh) was added to the inoculum, and the inoculum was filtered through cheesecloth and maintained on ice before use. A mock inoculation treatment that consisted of only phosphate buffer and carborundum was also used. Plants were inoculated an average of 30 d after seeding (when plants were approximately 9 cm in diameter). Inoculum was applied to the entire adaxial surface of the two newest leaves per plant using cotton-tipped applicators.


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  		Table 2. Potato virus Y (PVY) inoculation treatments used in research to investigate genetic resistance or tolerance in cv. K326 and derived isolines.

 
Plants were evaluated 21 d after inoculation for stem/veinal necrosis, interveinal necrosis, mottling, and leaf cupping/distortion. After evaluation for these symptoms, all plants were severed at the soil level. Fresh weights of the above-soil plant material were recorded to provide an indication of the degree of stunting caused by the PVY isolates.

Experimental Design
The inoculation experiment was conducted in a 3- by 3-m walk-in, controlled-environment growth chamber in the Southeastern Plant Environment Laboratory, Raleigh, NC (Thomas et al., 2006). A split-plot experimental design with three replications was used. The main-plot factor consisted of the virus inoculation treatment (Table 2), and the subplot factor consisted of the plant genotype (Table 1). Inoculation treatments were randomized within replications, and genotypes were randomized within inoculation treatments. The experiment was repeated three times. Experimental units were single plants grown in 600-mL round pots filled with a soil mix comprised of 50% river bottom sand and 50% Redi-earth Plug and Seedling Mix (Sun Gro Horticulture, Bellevue, WA). The chamber was maintained at 25 ± 0.5°C with an 18/6 h light/dark photoperiod. Plants were watered with a 0.5x standard nutrient solution (Thomas et al., 2006) once or twice daily, depending on the stage of plant growth.

Statistical Analyses
For the growth chamber experiment, an ANOVA appropriate for a split-plot design (McIntosh, 1983) was conducted using mixed model procedures facilitated by PROC MIXED of SAS (SAS Institute, Cary, NC). Main plots and subplots were considered as fixed factors and repetitions were considered as random. The ANOVA was used to test the main effects and their interactions on the measured characteristics. Main-plot, subplot, and main-plot x subplot means were generated using the LSMEANS statement. Mean separations were conducted using LSD tests at the 0.05 and 0.01 probability levels according to Steel et al. (1997).

Evaluating the Effects of the Nicotiana africana Genomic Region on Agronomic Traits
Plant Material and Experimental Design
It was of interest to know the influence, if any, of the introgressed N. africana genomic region on yield and quality when in hemizygous and homozygous conditions. Five Nafr/Nafr doubled haploid lines derived from PVY NSNR–tolerant K326 Nafr/— BC5F1 individuals were generated as described above. Each of these lines was also hybridized with K326 to produce five F1 hybrids hemizygous for Nafr. The five doubled haploid lines, the five corresponding Nafr/— F1 hybrids, and K326 were evaluated in the absence of PVY infection at four North Carolina locations during 2006: Central Crops Research Station (Clayton, NC), Upper Coastal Plain Research Station (Rocky Mount, NC), Oxford Tobacco Research Station (Oxford, NC), and the Lower Coastal Plain Tobacco Research Station (Kinston, NC). A randomized complete block design with four replications was used at each location. Each plot consisted of a single row with 22 competitive plants. Interrow spacing was 1.14, 1.16, 1.20, and 1.22 m at Clayton, Rocky Mount, Oxford, and Kinston, respectively. Within-row spacing was 0.56 m at all locations. The end plants of each plot served as guard plants and were removed before harvest. Transplants were produced in greenhouses using a float tray system, and experiments were transplanted to the field on 17 April, 19 April, 2 May, and 2 May at Rocky Mount, Kinston, Oxford, and Clayton, respectively. Suggested management practices for flue-cured tobacco production were used at each research station.

Leaves from all plants per row (excluding end plants, which were removed before harvest) were harvested in four separate harvests (primings) and flue cured. Each priming was weighed to generate yield data, and official USDA grades were assigned by a former USDA grader. A numerical reflection of cured leaf quality for each plot was generated using the 2006 North Carolina Flue-Cured Tobacco Grade Index (Smith and Fisher, 2007). Value per hundred weight (US$ cwt–1) was calculated based on average prices paid for standard grades during the 2006 growing season. Plot values for grade index and value per hundredweight were calculated using a weighted average across all four primings. Fifty-gram cured leaf samples were prepared for each plot by compositing cured leaf from each priming on a weighted-mean basis. Oven-dried samples were ground to pass through a 1-mm sieve and analyzed for total alkaloids and reducing sugars (expressed as a percentage of dry weight) using the method of Davis (1976).

Statistical Analyses
For the field experiment, a combined analysis of variance appropriate for analyzing a randomized complete block design across environments (McIntosh, 1983) was conducted using PROC MIXED. Entries and environments were considered as fixed and random factors, respectively. Entry means were produced using the LSMEANS statement and compared using calculated LSD values (Steel et al., 1997). Comparisons between K326, K326 Nafr/Nafr, and K326 Nafr/— group means were conducted using ESTIMATE statements in PROC MIXED.


   RESULTS
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Growth Chamber Experiment
Symptoms on inoculated plants in the growth chamber experiment were diverse, ranging from mild leaf mottling with little stunting to very severe systemic necrosis and plant death. Significant differences (P < 0.05) were observed between inoculation treatments and genotypes for all of the measured characters. Isolates having the greatest impact on overall plant growth (as reflected by reduced fresh weight) were those that induced severe necrotic effects, such as NSNR, MSNR, and VAM-B (Table 3 ). The isolates PVY MSMR, 99–04, YNW PL1, and YNW PL2 produced leaf mottling with little to no necrotic effects, and did not significantly reduce fresh weights relative to the mock inoculation treatment (Table 3).


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  		Table 3. Main-plot (inoculation treatment) means averaged across all genotypes.

 
Averaged across all inoculation treatments, six genotypes exhibited significantly greater fresh weight than K326 (P < 0.05) (Table 4 ). Genotype K326 Nafr/Nafr va/va produced the greatest fresh weight, although this was not significantly greater than the fresh weights for K326 va/va, K326 Nafr/— va/va, or K326 Nafr/— Va/va. Genotype K326 displayed significantly higher levels (P < 0.01) of stem/veinal necrosis relative to all other genotypes, and also displayed significantly higher levels (P < 0.01) of interveinal necrosis relative to all genotypes except K326 Va/va. Genotypes K326 Nafr/Nafr va/va and K326 Nafr/— va/va exhibited levels of leaf mottling that were significantly lower than that of all other genotypes (Table 4). Genotype K326 Nafr/Nafr exhibited higher levels of resistance to necrotic effects, leaf mottling, and stunting relative to K326 Nafr/—. Most of these differences were not statistically significant, however.


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  		Table 4. Subplot (genotype) means (averaged across inoculation treatments).

 
For many PVY isolates, symptom development was highly dependent on plant genotype. Comparisons of greatest interest are therefore those within specific inoculation treatments. Comparisons were made between genotypes within individual inoculation treatments using appropriate calculated LSD values (Table 5 ). Results from within-inoculation treatments are discussed below in the general order of increasing isolate severity.


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  		Table 5. Inoculation treatment (main plot) x cv. K326 genotype (subplot) means.

 
Potato Virus Y Isolates YNW PL1 and YNW PL2
Potato virus Y isolates YNW PL1 and YNW PL2 produced very similar symptoms in the growth chamber experiment and induced slight stem necrosis, mild leaf mottling, and slight leaf cupping on K326 and Va/va genotypes (Table 5). Significant differences between genotypes were observed only for leaf mottling and fresh weight. For both YNW PL1 and YNW PL2, va/va genotypes exhibited significantly lower levels of leaf mottling (P < 0.05) relative to K326 and K326 Nafr/Nafr. Interestingly, for both YNW PL1 and YNW PL2, K326 Va/va was not significantly different from K326 va/va for leaf mottling (Table 5). For YNW PL2, K326 Va/va exhibited significantly less mottling (P < 0.05) relative to K326. Also for YNW PL2, the fresh weights for K326 Nafr/Nafr and K326 Nafr/— va/va were significantly lower (P < 0.01) than those for K326 Va/va and K326 va/va. Neither K326 Nafr/Nafr nor K326 Nafr/— was significantly different from K326 for any of the evaluated characteristics

Potato Virus Y Isolate MSMR
Potato virus Y isolate MSMR did not induce necrotic effects on any genotype, but produced moderate levels of leaf mottling across all genotypes (Table 5). The degrees of leaf mottling displayed by K326 va/va and K326 Nafr/— va/va were significantly lower relative to all other genotypes (P < 0.05). Genotype K326 va/va exhibited significantly lower levels (P < 0.05) of leaf cupping/distortion relative to all other genotypes except K326 Nafr/— va/va. Neither K326 Nafr/Nafr nor K326 Nafr/— were significantly different from K326 for any of the measured symptoms.

Potato Virus Y Isolate 99-04
Slight stem necrosis and mild leaf mottling were observed for all genotypes inoculated with PVY isolate 99-04 (Table 5). Genotype K326 Nafr/Nafr va/va exhibited significantly lower levels (P < 0.05) of leaf mottling relative to K326 and K326 Nafr/—. Significant differences (P < 0.05) were also observed between genotypes for fresh weight, with the greatest fresh weight being produced by K326 va/va, and the lowest by K326 Nafr/Nafr. Homozygous recessive va/va genotypes appeared to provide some protection against symptoms produced by this isolate, while Nafr appeared to provide little additional benefit.

Potato Virus Y Isolate DL
Potato virus Y isolate DL produced moderate leaf mottling and leaf cupping/distortion on all genotypes, with little to no stem/veinal or interveinal necrosis (Table 5). Significant differences (P < 0.05) between genotypes were only identified for fresh weight, where K326 Nafr/— Va/va produced the greatest fresh weight, and K326 Nafr/— va/va the lowest.

Potato Virus Y Isolate Europe H
Potato virus Y isolate Europe H produced moderate stem/veinal necrosis and leaf mottling on K326 and Va/va genotypes (Table 5). All va/va genotypes exhibited significantly less stem/veinal necrosis than K326 and Va/va genotypes (P < 0.01). Genotypes of the va/va constitution exhibited lower levels of leaf mottling and leaf cupping/distortion relative to K326 and Va/va genotypes, although not all of these differences were statistically significant. Genotype K326 va/va Nafr/Nafr produced the highest fresh weight. The fresh weight for this genotype was significantly greater than the fresh weights for K326, K326 Nafr/Nafr, K326 Nafr/—, K326 Nafr/— Va/va, and K326 Va/va (P < 0.05). Genotype K326 Nafr/Nafr was not significantly different from K326 for any of the measured symptoms.

Potato Virus Y Isolate VAM-B
Potato virus Y isolate VAM-B produced slight necrotic effects in the stem and vascular tissue of K326, K326 va/va, and K326 Va/va. None was observed in any genotype containing Nafr, however (Table 5). Medium levels of interveinal necrosis, leaf mottling, and stunting (as reflected by lower fresh weights) were also exhibited by K326, K326 va/va, and K326 Va/va. Significantly lower levels of interveinal necrosis, leaf mottling, leaf cupping/distortion, and stunting were observed in Nafr-containing material relative to these three genotypes (P < 0.01). No significant differences were observed between K326 Nafr/Nafr and K326 Nafr/— for any characteristic.

Potato Virus Y Isolate NSNR
Potato virus Y isolate NSNR produced extremely severe necrotic effects on K326 and K326 Va/va. Genotypes possessing Nafr exhibited few necrotic effects in vascular tissues and were significantly resistant to these effects in comparison with K326 and K326 Va/va (P < 0.01) (Table 5). Genotype K326 va/va exhibited greater resistance to these necrotic effects than K326 (P < 0.01), but was not as resistant as materials carrying Nafr. Genotype K326 Nafr/Nafr va/va displayed significantly less leaf cupping or distortion relative to K326 Nafr/Nafr (P < 0.01). All genotypes produced significantly greater fresh weight than K326 and K326 Va/va (P < 0.01). Genotype K326 Nafr/Nafr was not significantly better than K326 Nafr/— for stem/veinal necrosis, but did exhibit significantly greater fresh weight and a significantly lower level of interveinal necrosis.

Potato Virus Y Isolate MSNR
All genotypes were affected by severe systemic necrosis after inoculation with PVY isolate MSNR (Table 5). Genotypes K326 and K326 Nafr/— exhibited significantly greater levels (P < 0.01) of stem/veinal and interveinal necrosis than all other genotypes. Genotype K326 Nafr/— va/va exhibited significantly lower levels (P < 0.05) of stem/veinal necrosis relative to all other entries. Genotype K326 Nafr/Nafr va/va exhibited significantly lower levels (P < 0.01) of interveinal necrosis relative to K326 va/va. In pairwise comparisons, fresh weights of va/va genotypes were significantly greater (P < 0.01) than fresh weights for K326 and Va/va genotypes.

Field Experiment
Five selected K326 Nafr/Nafr lines and five corresponding K326 Nafr/— F1 hybrids were evaluated for yield and quality characteristics in four North Carolina environments in the absence of PVY infection. Significant differences were detected among entries for yield, cash return (US$ ha–1), grade index, total alkaloids, and reducing sugars. Three entries were significantly lower yielding relative to K326 (P < 0.05), and one produced a significantly lower cash return (P < 0.05) (Table 6 ). No entry was significantly better than K326 for yield or cash return. Two entries had significantly higher total alkaloids than K326, and two lines had significantly greater reducing sugars relative to this check (P < 0.05) (Table 6). Compared with K326, three entries produced cured leaf with significantly better quality as reflected by grade index (P < 0.05) (Table 6). Two entries were significantly better for value per hundredweight (P < 0.05). No line exhibited cured leaf quality that was significantly lower than that of K326.


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  		Table 6. Entry means for K326 Nafr/Nafr lines and Nafr/— F1 hybrids evaluated in four North Carolina environments. Entries are ordered in terms of increasing cash return.

 
As a group, the K326 Nafr/Nafr doubled haploid lines were significantly lower yielding than K326 (197 kg ha–1, P < 0.05) (Table 7 ). Genotype K326 outyielded the Nafr/— F1 hybrids by 83 kg ha–1, but this difference was not statistically significant. As a group, the Nafr/— F1 hybrids produced significantly higher yields, a greater cash return, and higher reducing sugars than the group of Nafr/Nafr doubled haploid lines (P < 0.05) (Table 7). The Nafr/— F1 hybrid group also produced cured leaf with significantly better quality as reflected by grade index and value per hundredweight relative to K326 and the group of Nafr/Nafr doubled haploid lines.


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  		Table 7. Comparisons of trait means for K326 Nafr/Nafr lines, K326 Nafr/— F1 hybrids, and cv. K326 evaluated in four North Carolina environments.

 

   DISCUSSION
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Symptoms observed on plants inoculated with the nine PVY isolates were highly variable and often genotype dependent. No genotype exhibited complete resistance to any isolate. Relative to Nafr, the recessive va resistance mechanism had a tendency to provide greater resistance or tolerance against leaf mottling and leaf distortion caused by the more mild PVY isolates such as MSMR, YNW PL1, and YNW PL2. Relative to K326, va/va genotypes exhibited increased resistance against the necrotic effects of isolates Europe H, YNW PL1, YNW PL2, and NSNR. The va mechanism had little effect against necrosis induced by PVY isolate VAM-B, however. The introgressed Nafr region had little effect on leaf mottling caused by the mild isolates, but had very significant effects against the necrotic effects caused by severe isolates NSNR and VAM-B. The Nafr/Nafr genotypes exhibited much greater resistance to the necrotic effects caused by these two isolates than K326 va/va. Neither Nafr nor va was able to alleviate the severe systemic necrosis induced by PVY isolate MSNR, however. Genotype K326 is homozygous for the dominant gene Rk, which provides resistance against the root-knot nematode, Meloidogyne incognita. Development of systemic necrosis in tobacco genotypes carrying Rk is typical on inoculation with PVY MSNR (Burk et al., 1979b). Rufty et al. (1983) proposed that the necrotic response in Rk genotypes is due to pleiotropic effects of this resistance gene.

Breeding lines possessing Nafr may provide an additional source of genetic variability for developing tobacco cultivars with improved resistance to PVY. The results of this investigation demonstrate that va and Nafr can be combined into single genotypes to increase the range and level of resistance or tolerance to this economically important pathogen. Although slight resistance might be gained against certain effects caused by some PVY isolates in Va/va genotypes, it is generally assumed that homozygous recessive genotypes are necessary to obtain the greatest level of resistance from this mechanism. In this study, small differences were found between Nafr/Nafr and Nafr/— genotypes for resistance to PVY VAM-B. For PVY NSNR, however, Nafr/Nafr genotypes displayed lower levels of stunting and interveinal necrosis relative to Nafr/— hemizygotes.

When attention is given to yield and cash return, strong consideration might be given to deploying Nafr in the hemizygous condition. The yield penalty associated with Nafr was lessened when deployed in the hemizygous condition. Such breeding strategies are often used in tobacco (Wernsman and Rufty, 1987) and other species (Wehner, 1999) where disease resistance genes introgressed from wild relatives are frequently utilized. Interestingly, cured leaf quality as reflected by grade index and value per hundredweight was improved in materials possessing Nafr. Carlson (1995) also reported superior quality in materials possessing Nafr in the form of an intact addition chromosome. This is the first indication that germplasm introgressed from a wild Nicotiana relative might contribute to improved cured leaf quality in cultivated tobacco. Further data need to be collected to substantiate this finding.

Finally, it is important to note that the full level of resistance to PVY that has been reported for N. africana (Lucas et al., 1980) was not observed in the lines described here. It is not known if the full resistance expressed by this species is controlled by multiple genes on more than one chromosome of the N. africana genome, or if the resistance gene introgressed in our materials is influenced in a negative manner by modifying genes contained within the N. tabacum genome. Experiments are underway to better understand the genetics controlling the very high level of resistance exhibited by N. africana.


   ACKNOWLEDGMENTS
 
I am grateful to Philip Morris USA, Richmond, VA, for their support of the North Carolina State University tobacco breeding and genetics research program. I am also appreciative of assistance from Cara Rose and the staff of the Southeastern Environment Laboratory for maintenance of plant material. I also thank Dr. Jean-Louis Verrier and Dr. Teresa Doroszewska of Altadis (France) and Pulawy Institute for Soil and Plant Cultivation (Poland), respectively, for providing PVY isolates for this research.


   NOTES
TOP
 NOTES
ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
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Received for publication January 4, 2007.


   REFERENCES
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 





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HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
The SCI Journals Agronomy Journal Vadose Zone Journal
Journal of Natural Resources
and Life Sciences Education		 Soil Science Society of America Journal
Journal of Plant Registrations Journal of
Environmental Quality		 The Plant Genome