Published online 1 February 2006
Published in Crop Sci 46:554-560 (2006)
© 2006 Crop Science Society of America
677 S. Segoe Rd., Madison, WI 53711 USA
PLANT GENETIC RESOURCES
Resistance to Tobacco Black Shank in Nicotiana Species
B.-C. Lia,*,
W. T. Bassb and
P. L. Corneliusc
a Kentucky Tobacco Research and Development Center, University of Kentucky, Lexington, KY 40546
b ARS-USDA, Mid South Area Forage-Animal Production Research Unit
c Department of Agronomy, University of Kentucky, Lexington, KY 40546
* Corresponding author (bli2{at}uky.edu)
 |
ABSTRACT
|
|---|
Black shank [caused by Phytophthora parasitica (Dast.) var. nicotianae (B. de Haan) Tucker] is one of the most devastating diseases in tobacco (Nicotiana tabacum L.). Phytophthora parasitica var. nicotianae race 1 is the most damaging race of this fungus. The objective of this study was to identify the Nicotiana species that are resistant to P. parasitica var. nicotianae race 1 as part of the effort to develop Nicotiana-based plant varieties for applications in the production of plant-made pharmaceuticals (PMPs). The roots of approximately 26-d-old growth room–grown plants were inoculated with zoospores of an isolate of P. parasitica var. nicotianae race 1 by a dipping procedure. The percentage of plants that were free of symptoms 10 to 14 d after inoculation was taken as a measure of resistance to the fungus. We evaluated a total of 97 Nicotiana accessions from 37 species from the USDA collection and other sources. Among the 97 accessions, three from N. debneyi Domin, two from N. repanda Willd. ex Lehm., and one each from N. megalosiphon Van Heurck & Mull. Arg., N. plumbaginifolia Viv., N. suaveolens Lehm., and N. sylvestris Speg. & Comes were found to be resistant. Their resistances were further confirmed after they were evaluated for an additional three times. These nine accessions were then challenged by another isolate of race 1. All accessions except for the N. sylvestris accession were found to be resistant. Nicotiana debneyi, N. megalosiphon, and N. suaveolens were not previously reported to be resistant to P. parasitica var. nicotianae race 1. Our study indicated that the undomesticated Nicotiana species constitute a rich genetic source for resistance to the tobacco disease black shank.
Abbreviations: PMP, plant-made pharmaceutical
|
INTRODUCTION
|
|---|
TOBACCO EXHIBITS several attributes that make it attractive for the production of PMPs, including its compatibility for high-level expression systems for foreign proteins, high biomass production, and nonfood status (Daniell et al., 2001; Giddings et al., 2000; Maliga, 2003). However, traditional tobacco varieties are not ideal for these pharmaceutical applications, since many lack resistance to major diseases and are not suitable for direct seeding, identity preservation, and multiple harvests. We aim to develop a Nicotiana-based, fully optimized new "vehicle" plant for PMP production that will incorporate these additional traits.
Nicotiana germplasm includes species with important traits for PMP-oriented plant variety development, such as high biomass production (Mundell and Chambers, personal communication, July 2004), high shoot-organogenic potential (Li et al., 2003), resistance to major tobacco diseases (Clayton, 1945; Heist et al., 2004; Litton et al., 1970a), tolerance to abiotic stresses such as salt and drought (Komori et al., 2000), and varying levels of alkaloid content (Saitoh et al., 1985). As part of the effort to develop new plant varieties for PMP applications, we have examined diverse Nicotiana accessions for their responses to Phytophthora parasitica var. nicotianae, the causal agent of tobacco black shank, one of the most important diseases in tobacco (Collins et al., 1971; Csinos, 1999).
Phytophthora parasitica var. nicotianae, a soilborne pathogen that can cause a serious disease to roots and stems of tobacco, is distributed throughout the United States and in many tobacco-growing regions of the world. Any cultivar developed for PMP applications should be resistant to the fungus. Phytophthora parasitica var. nicotianae includes two major races, race 0 and race 1 (Stokes and Litton, 1966). Resistance to race 0, identified in N. longiflora Cav. and N. plumbaginifolia, is controlled by a single dominant gene, and breeding efforts to develop cultivars highly resistant to race 0 have been successful, but there are no commercial cultivars highly resistant to race 1 (Valleau et al., 1960; Collins et al., 1971; Csinos, 1999). Therefore, identification of Nicotiana species with very high levels of resistance to race 1 should greatly aid efforts to develop cultivars with high levels of resistance to race 0 and race 1.
Efforts were made to identify resistance to race 1 in Nicotiana species in a previous study (Litton et al., 1970a). Fifty-seven Nicotiana species were examined, and six were found to be resistant. They were N. longiflora, N. nudicaulis S. Watson, N. plumbaginifolia, N. repanda, N. stocktonii Brandegee, and N. rustica L. However, that study only examined one accession for most of the 57 species. Some Nicotiana species resistant to race 1 might not have been identified, as accessions could vary in their response to infection. Also, there are a total of sixty-seven documented Nicotiana species (Anon, 1990; Goodspeed, 1954; Laskowska and Berbec, 2003), so some Nicotiana species have never been examined for their responses to P. parasitica var. nicotianae race 1.
In this study, we evaluated 97 Nicotiana accessions from 37 species for their resistance to two isolates of P. parasitica var. nicotianae race 1, Isolate 1 and Isolate 2. The objective of this study was to identify the Nicotiana species that are resistant to P. parasitica var. nicotianae race 1. The resistance was measured in terms of the percentage of plants that did not show any symptoms 10 to 14 d after inoculation. Nine accessions, three from N. debneyi, two from N. repanda, and one each from N. megalosiphon, N, plumbaginifolia, N. suaveolens, and N. sylvestris were found to be resistant to Isolate 1. All these accessions, except for the N. sylvestris accession, were also resistant to Isolate 2.
|
MATERIALS AND METHODS
|
|---|
Evaluation of Nicotiana species for their responses to P. parasitica var. nicotianae was performed using the small-plant technique (Litton et al., 1970b). All experiments were conducted in a growth room maintained at 22 to 23°C with a 12-h photoperiod and a light intensity of approximately 112 µmol m–2 s–1. Two isolates of P. parasitica var. nicotianae race 1, Isolate 1 and Isolate 2, were used in this study and they were both collected from local tobacco fields in Kentucky that were infected by the fungus. These two isolates have been used by local breeders to screen their breeding materials for resistance to black shank and were considered to be typical to the Kentucky types. Pathogen culture and inoculum preparation were performed as described previously (Litton et al., 1970b).
Approximately 10 seeds were sown on the surface of moist vermiculite in 50-mL plastic tubes suspended through holes bored in metal racks. The tubes had a 6-mm hole in the bottom through which nutrient solution and inoculum were introduced by a dipping procedure. Twenty-six days after seeding, the roots of the plants were inoculated by submerging the tubes in a suspension of active zoospores of P. parasitica var. nicotianae in a one-third Hoagland's solution (Litton et al., 1970b).
After inoculation the racks of tubes were placed across the top of temperature-controlled tanks with the bottom of the tubes about 1 inch above the surface of the water. A small-bladed electronic stirrer was mounted on each tank to circulate the water to maintain a uniform temperature at the surface. An immersion-type heating coil was used in each tank to heat the water so that a temperature of 31 to 32°C was maintained in the root zone of the plants above the water (Litton et al., 1970b).
The rating was made 10 to 14 d after inoculation with an isolate of P. parasitica var. nicotianae race 1, when the susceptible control plants were dead or almost dead. The root systems were washed free of vermiculite and each individual root was examined. Plants that were alive and free of obvious symptoms in their roots were classified as healthy or resistant, and those that had symptoms as susceptible. The susceptible plants include those that were either dead (Fig. 1A
), or alive but having obvious root system symptoms, with single or multiple roots being necrotic (Fig. 1B). The percentage of inoculated plants that were healthy was used to measure the resistance. Three tobacco cultivars, Beinhart, KY14, and KY14XL8, were used as experimental controls in each evaluation. Beinhart is resistant to race 1 and race 0 (Litton et al., 1970a), KY 14 is susceptible to both race 1 and race 0, and KY14XL8 is resistant to race 0, but susceptible to race 1. The resistance of KY14XL8 to race 0 came from burly tobacco line L8, which contains the resistant factor from N. longiflora (Valleau et al., 1960). L8 is highly resistant to race 0, but susceptible to race 1 (Valleau et al., 1960; Stokes and Litton, 1966). Those accessions that yielded a similar or higher percentage of healthy plants compared with the resistant control Beinhart were regarded as resistant, and those that yielded a lower or zero percentage of healthy plants were regarded susceptible.
View larger version (130K):
[in this window]
[in a new window]
|
Fig. 1. Responses of Nicotiana species to Phytophthora parasitica var. nicotianae race 1, 10 to 14 d after inoculation. (A) Some plants were dead and some alive; (B) The roots of the alive plants were symptomatic, with either single (left) or multiple (right) roots being necrotic.
|
|
We started with the evaluation of 97 Nicotiana accessions against Isolate 1 of P. parasitica var. nicotianae race 1 and each accession was evaluated twice, with three or four replications per evaluation. Each plastic tube with approximately 10 plants represented one replication. Those accessions that yielded a similar or higher percentage of healthy plants, compared with Beinhart, were selected and evaluated three additional times against the same Isolate 1, each with four replications. They were then also evaluated three times against Isolate 2, each also with four replications. All experiments were performed as a randomized complete block design.
Data from all the experiments were pooled for statistical analysis and reported here. The percentage of plants that were free of disease was analyzed using a generalized linear model assuming a binomial distribution and using a logit link function (McCullagh and Nelder, 1989; Dobson, 1990). Computation was by SAS Procedure Genmod (SAS Institute Inc., 1999), fitting a model consisting of a constant term (intercept) plus "accession" effects. The "dscale" option was used to allow for overdispersion, that is, the scale parameter was estimated as (deviance/f)
; where f is the degrees of freedom (df) of the deviance. ("Deviance" is twice the difference between logarithms of maximized likelihoods of a saturated model and the fitted model.) Some accessions did not have any healthy plants, and, because the Procedure Genmod solution would not converge if these accessions were included, these were omitted from the analysis. Even if they could be included, their inclusion would result in a downward biased estimate of the scale parameter for purposes of testing hypotheses concerning differences among accessions that did have healthy plants.
Overdispersion was statistically very highly significant (P < 0.0001) in all three experiments, significance here being determined by comparing the deviance with the chi-square distribution with f df. An "lsmeans" statement with "diff" option was used to make pairwise comparisons of "accessions." A Wald chi-square test is the only test of pairwise differences that Procedure Genmod computes. We recomputed the P values for the differences regarding the chi-square values as F statistics with one numerator df and f denominator df. Because f was rather large for all three experiments, this resulted in rather trivial increases in P values. Back-transformation of the lsmeans from the logit scale to binomial probabilities confirmed that the lsmeans were equivalent to logits of the empirical binomial proportions for the various accessions.
|
RESULTS
|
|---|
We started with Nicotiana germplasm comprising 123 accessions from 52 species. Among them, however, 8 accessions from 7 species did not germinate, 18 accessions from 14 species germinated late, and all these 26 accessions were dropped from the evaluation due to scheduling conflicts. Therefore, 97 Nicotiana accessions, representing 37 species, were evaluated for their responses to Isolate 1 of P. parasitica var. nicotianae race 1 in this study, and 22 of the 37 species included more than one accessions (Table 1).
View this table:
[in this window]
[in a new window]
|
Table 1. Responses of Nicotiana species to Isolate 1 of Phytophthora parasitica var. nicotianae race 1, as measured by the percentage of healthy plants 10 to 14 d after inoculation.
|
|
Statistical analysis on the pooled data from two repeated evaluations for the responses of the 97 Nicotiana accessions to Isolate 1 of P. parasitica var. nicotianae race 1 identified significant differences among the 97 Nicotiana accessions with regard to their percentage of healthy plants (Table 1). Compared with Beinhart, a tobacco cultivar that is resistant to P. parasitica var. nicotianae race 1, nine accessions from six species yielded either a similar or higher percentage of healthy plants. These nine accessions were N. debneyi PI 503320, PI 503323, and AusTRC 303706 (accession number from the collection of Australian Tobacco Research Center); N. megalosiphon PI 555536; N. plumbaginifolia PI 302478; N. repanda PI 555551 and PI 555552; N. suaveolens Aus TRC 303709; and N. sylvestris S-8–1 (where the S number represents the collection number of the Kentucky Tobacco Research and Development Center [KTRDC]). Thirty-nine accessions from 23 species and the two susceptible control cultivars, KY14XL8 and KY14, yielded a significantly lower percentage of healthy plants, ranging from 1 to 32%; forty-nine accessions from 24 species did not yield any healthy plants.
The nine accessions that yielded a similar or higher percentage of plants that were free of symptoms were selected and reevaluated for an additional three times against Isolate 1 to confirm their resistance to the fungus. Statistical analysis using the pooled data from the three repeated evaluations indicated that all nine accessions yielded either a similar or a higher percentage of plants that were resistant, comparable to the resistant cultivar Beinhart. Among them, four accessions, N. debneyi PI 503320, N. repanda PI 555551 and PI 555552, and N. sylvestris S-8–1, yielded significantly higher percentages, ranging from 61 to 95%; five accessions, N. debneyi PI 503323 and AusTRC 303706, N. megalosiphon PI 555536, N. plumbaginifolia PI 302478, and N. suaveolens Aus TRC 303709, yielded similar percentages, ranging from 43 to 51%. Two susceptible control cultivars, KY 14 and KY14XL8, yielded significantly lower percentages, ranging from 1 to 9%.
These nine accessions were then evaluated three times against Isolate 2 of P. parasitica var. nicotianae race 1 (Table 2). Statistical analysis using the pooled data from the repeated evaluations identified significant differences among the nine accessions with regard to their responses to Isolate 2. Compared with the resistant cultivar Beinhart, three accessions, N. debneyi PI 503323 and N. repanda PI 555551 and PI 555552, yielded higher percentages of plants that were free of symptoms, ranging from 63 to 83%; five accessions, N. debneyi PI 503320 and AusTRC 303706, N. megalosiphon PI 555536, N. plumbaginifolia PI 302478, and N. suaveolens Aus TRC 303709, yielded similar percentages, ranging from 19 to 61%. One accession, N. sylvestris S-8–1, yielded a significantly lower percentage of healthy plants compared with Beinhart, only 5%, and was regarded as susceptible to this isolate. Two susceptible control cultivars, KY14 and KY14XL8, also yielded significantly lower percentages, both 2%.
View this table:
[in this window]
[in a new window]
|
Table 2. Responses of selected Nicotiana species to Isolate 1 and Isolate 2 of Phytophthora parasitica var. nicotianae race 1, as measured by the percentage of healthy plants 10–14 days after inoculation.
|
|
|
DISCUSSION
|
|---|
To identify Nicotiana species that are resistant to P. parasitica var. nicotianae race 1, this study evaluated the responses of 97 Nicotiana accessions from 37 species to two isolates of P. parasitica var. nicotianae race 1. We found that three accessions from N. debneyi, two from N. repanda, and one each from N. megalosiphon, N. plumbaginifolia, and N. suaveolens were resistant to both Isolate 1 and Isolate 2, and an N. sylvestris accession was resistant to Isolate 1 but susceptible to Isolate 2. Nicotiana debneyi, N. megalosiphon, and N. suaveolens have not been reported previously to be resistant to P. parasitica var. nicotianae race 1, although they were evaluated in the previous study (Litton et al., 1970a). This discrepancy could result from the lower number of accessions evaluated for each of the three species in the previous study. Only one accession was evaluated previously from N. debneyi, one from N. megalosiphon, and two accessions from N. suaveolens, whereas six N. debneyi accessions, two N. megalosiphon accessions, and seven N. suaveolens accessions were evaluated in the present study.
Nicotiana longiflora, N. nudicaulis, and N. rustica were classified to be resistant in the previous study but were found to be susceptible in this study. This difference could result from the different accessions evaluated in the two studies, or differences in the isolates used. Nicotiana plumbaginifolia and N. repanda were resistant in both studies; N. stocktonii was resistant in the previous study but was not evaluated in this study because of its late germination. Two species, N. kawakamii Y. Ohashi and N. africana Merxm., that were not evaluated previously, were found not to be resistant in this study. Taking the results from this study and the previous study together, a total of nine Nicotiana species have been identified to be resistant to Phytophthora parasitica var. nicotianae race 1, namely N. debneyi, N. longiflora, N. megalosiphon, N. plumbaginifolia, N. repanda, N. nudicaulis, N. rustica, N. stocktonii, and N. suaveolens.
The small-plant technique used in this study was originally developed by Stokes and Litton (1966) and later modified by Litton et al. (1970b). It has been used as a standard technique for evaluating the response of Nicotiana species including tobacco to P. parasitica var. nicotianae (Litton et al., 1970a). The main advantage of this technique is that it allows the screening of a large number of candidate plants in a short period of time. Also, the fact that only small seedlings were used in this technique makes it a very stringent method for screening plant materials for resistance to P. parasitica var. nicotianae. Plant materials that were identified to be resistant using this technique are more than likely to be more resistant when they become older, since it has been well established that older plants are generally more resistant to plant pathogens than those that are younger. Tobacco plants are typically subjected to P. parasitica var. nicotianae only when they become older, after being transplanted in the field.
Plants in Nicotiana species are self-pollinated, and their seeds are generally genetically homogeneous (Goodspeed, 1954). One question that has arisen from this study and the previous study, though, is why none of the resistant Nicotiana species gave 100% resistance against P. parasitica var. nicotianae race 1, while some Nicotiana species like N. longiflora and N. plumbaginifolia gave 100% resistance against P. parasitica var. nicotianae race 0 (Litton et al., 1970a). The resistance of N. longiflora and N. plumbaginifolia to race 0 has been identified to be a qualitative trait, conditioned by a single dominant gene (Collins et al., 1971), but the mode of inheritance for the resistance of Nicotiana species to race 1 has not been elucidated. We speculate that the resistance to P. parasitica var. nicotianae race 1 in the Nicotiana species identified in this study and in the previous study is likely to be a quantitative trait, conditioned by multiple genes. The resistance of qualitative nature, or in a gene-for-gene fashion (Flor, 1971), is known to be generally strong and highly effective, but the quantitative resistance tends to be weak (Vanderplank, 1963). A plant population for any Nicotiana accession, though genetically homogeneous, is not likely to be a uniform one, and the individual plants may vary in their growth and development because of environmental and physiological reasons. This variation may result in variation in their response to P. parasitica var. nicotianae. Some individuals in a population of quantitative resistance may be susceptible to infection by P. parasitica var. nicotianae and produce symptoms, and others in the same population may not. This may apply to the Nicotiana species that were identified in this study and in the previous study to be resistant to P. parasitica var. nicotianae race 1. On the other hand, the individuals in a population of qualitative resistance may be all highly resistant, though varied among them, that none of them got infected. This may apply to the resistance of N. longiflora and N. plumbaginifolia to P. parasitica var. nicotianae race 0, which was identified in the previous study (Litton et al., 1970a). At any rate, the yielding of less-than-100% resistance in Nicotiana species against race 1 is unlikely due to the small plant technique used in this study and in the previous study, since a similar result was also obtained for N. tabacum and their hybrids using older plants that were grown in an aluminum tray (Apple, 1963).
There appears to be a relationship between resistance to P. parasitica var. nicotianae race 1 and phylogenetic groups in Nicotiana species. The nine Nicotiana species that are reported to be resistant to P. parasitica var. nicotianae race 1 in this study, and in the previous study, were clustered in three of the eight branches in a phylogenetic Nicotiana tree based on the nucleotide sequence of the matK gene (maturase K, a chloroplast gene) (Aoki and Ito, 2000). However, there appears to be no relationship between resistance to P. parasitica var. nicotianae race 1 and native habitats. All nine Nicotiana species that have been identified to be resistant to P. parasitica var. nicotianae race 1 were distributed in all three major habitats, South America, North America, and Australia (Table 1). This is in contrast with the resistance to Peronospora tabacina Adam, the causal agent of blue mold in tobacco, where most of the Nicotiana species that were resistant were distributed in Australia (Clayton, 1945).
The differential responses of N. debneyi PI 503323 and N. sylvestris S-8–1 to Isolate 1 and Isolate 2 of P. parasitica var. nicotianae race 1 in the re-evaluation of the nine selected Nicotiana accessions (Table 2) suggested that the two isolates differed in their pathogenicity. Compared with the resistant cultivar Beinhart, the percentage of healthy N. debneyi PI 503323 plants was not significantly different when inoculated with Isolate 1, but was significantly higher when inoculated with Isolate 2. As for N. sylvestris S-8–1, that percentage was significantly higher when inoculated with Isolate 1 and significantly lower when inoculated with Isolate 2.
In summary, this study demonstrated that five Nicotiana species were resistant to Isolate 1 and Isolate 2 of P. parasitica var. nicotianae race 1. Of those five species, three, N. debneyi, N. megalosiphon and N. suaveolens, were not previously reported to be resistant to P. parasitica var. nicotianae race 1. A total of nine Nicotiana species have been identified to be resistant to P. parasitica var. nicotianae race 1. Therefore, the potential use of undomesticated Nicotiana species as a source for genetic resistance to the tobacco disease black shank is suggested.
|
ACKNOWLEDGMENTS
|
|---|
We acknowledge with appreciation the contribution of Dr. William Nesmith, Extension Plant pathologist Emeritus, University of Kentucky, to this work, including his frequent counsel, supplying the Isolate 2, and critical review of the manuscript. We also want to thank Dr. Maelor Davies for his support, discussions and the review of the manuscript, Drs. Verne Sisson, Robert Miller, and Milton Zaitlin for kindly providing us the seeds for Nicotiana species, Brenda Kennedy for discussions and providing us the Isolate 1, and Rich Mundell, Dr. Orlando Chambers, Peggy Rice, Bonnie Kinney, and Chris Cooper for collecting and/or reproducing Nicotiana seeds or watering the plants. Funding from Kentucky Tobacco Research Board.
Received for publication April 25, 2005.
|
REFERENCES
|
|---|
- Anon. 1990. Illustrated book of the Genus Nicotiana. Japan Tobacco Inc. Plant Breeding and Genet. Res. Lab., Tokyo.
- Aoki, S., and M. Ito. 2000. Molecular phylogeny of Nicotiana (Solanaceae) based on the nucleotide sequence of the matk gene. Plant Biol. 2:316–324.[CrossRef]
- Apple, J.L. 1963. Physiological specialization within Phytophthora parasitica var. Nicotianae. Phytopathology 52:351–354.
- Clayton, E.E. 1945. Resistance of tobacco to blue mold (Peronospora tabacina). J. Agric. Res. 70:79–87.
- Collins, G.B., P.D. Legg, C.C. Litton, and M.J. Kaserbauer. 1971. Inheritance of resistance to black shank in Nicotiana tabacum L. Can. J. Genet. Cytol. 13:422–428.
- Csinos, A.S. 1999. Stem and root resistance to tobacco black shank. Plant Dis. 83:777–780.
- Dobson, A.J. 1990. An introduction to generalized linear models. Chapman and Hall, London.
- Daniell, H., S.J. Streatfield, and K. Wykoff. 2001. Medical molecular farming: Production of antibodies, biopharmaceuticals and edible vaccines in plants. Trends Plant Sci. 5:219–226.
- Flor, H.H. 1971. Current status of the gene-for-gene concepts. Annu. Rev. Phytopathol. 9:275–296.[CrossRef][ISI]
- Giddings, G., G. Allison, D. Brooks, and A. Carter. 2000. Transgenic plants as factories for biopharmaceuticals. Nat. Biotechnol. 18:1151–1155.[CrossRef][ISI][Medline]
- Goodspeed, T.H. 1954. The Genus Nicotiana: Origins, relationships and evolution of its species in the light of their distribution, morphology and cytogenetics. Chronica Botanica, Waltham, MA.
- Heist, E.P., D. Zaitlin, D.L. Funnell, W.C. Nesmith, and C.L. Schardl. 2004. Necrotic lesion resistance induced by Peronospora tabacina on leaves of Nicotiana obtusifolia. Phytopathology 94:1178–1188.
- Komori, T., P.N. Myers, S. Yamada, T. Kubo, and H. Imaseki. 2000. Comparative study of the Nicotiana species with respect to water deficit tolerance during early growth. Euphytica 116:121–130.[CrossRef]
- Laskowska, D., and A. Berbec. 2003. Preliminary study of the newly discovered tobacco species Nicotiana wuttkei Clarkson et Symon. Genet. Resour. Crop Evol. 50:835–839.[CrossRef]
- Li, B.-C., W. Huang, and T. Bass. 2003. Shoot production per responsive leaf explant increases exponentially with explant organogenic potential in Nicotiana species. Plant Cell Rep. 22:231–238.[Medline]
- Litton, C.C., G.B. Collins, and P.D. Legg. 1970a. Reaction of Nicotiana tabacum and other Nicotiana species to race 0 and 1 of Phytophthora parasitica var. nicotianae. Tobacco Sci. 14:144–146.
- Litton, C.C., G.B. Collins, and P.D. Legg. 1970b. A greenhouse technique for screening tobacco seedlings for black shank resistance. Tobacco Sci. 14:124–125.
- Maliga, P. 2003. Progress towards commercialization of plastid transformation technology. Trends Biotechnol. 21:20–28.[CrossRef][ISI][Medline]
- McCullagh, P., and J.A. Nelder. 1989. Generalized linear models. 2nd ed. Chapman and Hall, London.
- Saitoh, F., M. Noma, and N. Kawashima. 1985. The alkaloid contents of sixty Nicotiana species. Phytochemistry 24:477–480.[CrossRef]
- SAS Institute Inc. 1999. SAS/STAT user's guide. Version 8. SAS Institute Inc., Cary, NC.
- Stokes, G.W., and C.C. Litton. 1966. Source of black shank resistance in tobacco and host reaction to races 0 and 1 of Phytophthora parasitica var. nicotianae. Phytopathology 56:678–680.
- Valleau, W.D., G.W. Stokes, and E.M. Johnson. 1960. Nine years experience with the Nicotiana longiflora factor for resistance to Phytophthora parasitica var. nicotianae in the control of black shank. Tobacco Sci. 4:92–94.
- Vanderplank, J.E. 1963. Plant diseases: Epidemics and control. Academic Press, New York.
TOP
ABSTRACT
INTRODUCTION
