© 2008 Plant Management Network.
Clarifying the Source of Black Shank Resistance in Flue-cured Tobacco
Charles S. Johnson, Professor, Jeremy A. Pattison, Assistant Professor, and Elizabeth M. Clevinger, Research Specialist Senior, Southern Piedmont Agricultural Research and Extension Center, Virginia Polytechnic Institute and State University, Blackstone 23824; Thomas A. Melton, Assistant Director and Associate State Program Leader ANR/CRD, North Carolina Cooperative Extension, North Carolina State University, Raleigh 27695; Bruce A. Fortnum, Professor, Clemson University, Pee Dee Research and Education Center, Florence, SC 29506; and Asamina Mila, Assistant Professor and Extension Specialist, Department of Plant Pathology, North Carolina State University, Raleigh 27695
Johnson, C. S., Pattison, J. A., Clevinger, E. M., Melton, T. A., Fortnum, B. A., and Mila, A. 2008. Clarifying the source of black shank resistance in flue-cured tobacco. Online. Plant Health Progress doi:10.1094/PHP-2008-0618-02-RS.
Widespread use of resistance to race 0 of Phytophthora nicotianae in flue-cured tobacco (Nicotiana tabacum) has increased problems with race 1 in commercial fields. The RAPD marker UBC30, tightly linked to the Ph gene for resistance to race 0, was used to clarify the presence of the Ph gene in specific cultivars to enable farmers to more appropriately match cultivar resistance to the pathogen races predominating in their fields. The marker UBC30 was present in 20 of the 31 flue-cured tobacco cultivars tested, including CC 27, GL 350, NC 196, SP 220, SP 225, SP 227, and NC 810. These cultivars were previously thought to not possess the Ph gene. Presence of UBC30 was highly correlated (r = 0.93; P ≤ 0.001) with survival in fields infested primarily with race 0, and with greater survival in fields infested primarily with race 0 versus race 1 of the pathogen (r = 0.76; P ≤ 0.001). The likely presence of the Ph gene in so many currently grown flue-cured tobacco cultivars may limit farmers’ ability to shift pathogen populations back to race 0 from race 1 via the recommended cultivar rotation strategy.
Black shank is the common name for a root and stem rot that is among the most widespread and damaging tobacco diseases all over the world (6). Management of tobacco black shank has relied upon a combination of crop rotation, fungicides, and host resistance (15). Black shank resistance has been incorporated into cultivated tobacco from three sources, a Nicotiana tabacum breeding line Florida 301, N. longiflora, and N. plumbaginifolia (10,16). Resistance from N. longiflora and N. plumbaginifolia involve single, dominant genes that are highly effective against race 0 of the black shank pathogen but provide no protection against race 1 (1,2). Resistance derived from Florida 301 was the preferred source of black shank resistance for approximately 40 years because it is effective against both pathogenic races thought to predominate in the United States (0 and 1) (2,18). Unfortunately, resistance derived from FL 301 has only provided moderate protection against black shank.
Beginning in 1996, the black shank resistance available to farmers dramatically improved with the release of a series of flue-cured tobacco hybrids possessing the Ph gene, originally derived from N. plumbaginifolia (3,9). Estimated use of soil fungicides in North Carolina dropped from approximately 74% of planted tobacco hectares in 1987 to 15% in 2006 as planting of these hybrid cultivars increased from 1% to 47% (12,13). Unfortunately, several recent reports have related increased occurrence of race 1 of the black shank pathogen and subsequent increased disease losses with the widespread planting of these cultivars (Fig. 1) (4,17). Estimated incidence of P. nicotianae race 1 in Georgia tobacco fields increased from 16% prior to the release of these hybrids to 83% in 2003 (4,5).
Unfortunately, the presence or absence of the Ph gene in specific tobacco cultivars currently available to growers has become increasingly unclear, largely because the pathogen population structure in black shank resistance "nurseries" is now a largely unknown mixture of races, and the specific race present in each field plot cannot be known. However, randomly amplified polymorphic DNA (RAPD) markers linked to the Ph gene are available, and the work reported here determined the presence or absence of the gene in tobacco cultivars available to farmers in 2006 and 2007 using a coupling phase RAPD marker generated by the University of British Columbia and Operon primer sequence UBC30490 (5’ CCG GCC TTA G 3’) (8). Laboratory results were correlated with data from field experiments comparing survival of flue-cured tobacco germplasm in fields infested with the black shank pathogen.
DNA Extraction and RAPD Amplifications
A collection of diverse hybrid and inbred tobacco breeding lines and cultivars were greenhouse propagated (Table 1) from seed. Young leaves were collected for DNA extraction and stored at —60°C. DNA was extracted using the GenElute Plant Genomic DNA Miniprep Kit (Sigma-Aldrich, St. Louis, MO) according to the manufacturer’s instructions. PCR was performed in 25-µl reactions in a Bio-Rad iCycler thermocycler (Bio-Rad Laboratories Inc., Hercules, CA). Reaction conditions consisted of 50 ng of genomic DNA, 0.4 µM of primer, 1X green GoTaq reaction buffer (Promega, Madison, WI), 2.5 mM MgCl2, 200 µM each of dATP, dGTP, dTTP, and dCTP, and 1.0 U of GoTaq DNA polymerase (Promega, Madison, WI). The amplification profile used was modified from Johnson et al (8). The reaction was initially denatured at 94°C for 2 min, followed by 3 cycles at 94°C for 1 min, 38°C for 1 min, and 72°C for 2 min. An additional 35 cycles at 92°C for 1 min, 40°C for 1 min, 72°C for 2 min, and a final extension step of 72°C for 5 min. Nine microliters of each sample was loaded on a 2% agarose gel and separated at 110 volts for 2 h in 0.5X Tris-Borate-EDTA. DNA was visualized using ethidium bromide and fragment size estimated from a 1 kb DNA ladder (Promega, Madison, WI). The expected band size for the linked fragment was approximately 490 base pairs (8) (Fig. 2).
Table 1. Flue-cured tobacco cultivars screened for the presence of RAPD markers linked to black shank resistance from the Ph gene.
Disease Data Collection
Historical data was sought that might indicate relative survival of currently available flue-cured tobacco cultivars when either race of P. nicotianae may have predominated. Annual field data on black shank resistance collected (as part of a regional cultivar evaluation program) since 1996 by North Carolina State University and Clemson University field tests were used to estimate resistance to race 0 or 1 of P. nicotianae. Tobacco introduction 1071 (TI 1071) had been included in these tests to indicate the relative predominance of race 1 versus race 0 in each experiment (2). Race 0 was assumed to have predominated when the final percent survival of TI 1071 was high (greater than 80%), whereas race 1 was assumed to predominate when survival of TI 1071 was low (less than 50%). Survival of standard susceptible flue-cured tobacco cultivar K 326 in each experiment indicated the relative black shank disease pressure compared to all other tests. Disease incidence data for race 0 was available from nine field experiments conducted at seven research stations or farms from 1999 to 2004. Final survival of TI 1071 averaged 96%, compared to 44% for K 326. Disease incidence data for race 1 was obtained from nine field experiments at five research stations or farms, and conducted in 2000 and each year from 2003 to 2006. Percent survival of TI 1071 in the race 1 experiments averaged only 8% compared to 31% on K 326.
Flue-cured Tobacco Cultivars With or Without a Marker Linked to the Ph Gene
The results of our molecular marker analysis illustrate the difficulty in discriminating between single gene, vertical resistance and multiple gene, horizontal resistance based solely on field data. Presence of the polymorphism for coupling phase RAPD marker UBC30 indicated that 20 of the 31 flue-cured tobacco cultivars tested possess the Ph gene for resistance to tobacco black shank (Fig. 2), although seven of these (CC 27, GL 350, NC 196, SP 220, SP 225, SP 227, and NC 810) have been previously reported to only possess the horizontal black shank resistance derived from FL 301 (7,11). With the exception of SP 225 and SP 234, origin of the Ph gene in those cultivars where its presence was previously unreported is unclear from the reported pedigrees (Table 1).
Correlation Between Molecular Marker for Resistance and Field Performance
Presence of the polymorphism was highly correlated with cultivar survival in fields primarily infested with race 0 of P. nicotianae (r = 0.94; P ≤ 0.001) and with differential resistance to races 0 and 1 of the pathogen (r = 0.76; P ≤ 0.001) (Table 2). Cultivars possessing the polymorphism for UBC30 averaged approximately 42% (10% to 68%) higher survival against race 0 than against race 1, compared to a mean 8% (1% to 27%) greater survival against race 0 versus race 1 among cultivars without the polymorphism (Table 2). The ranges in survival may have resulted from differing levels of quantitative resistance to black shank, originally obtained from Florida 301, among the cultivars tested.
Table 2. Reported presence of the Ph gene, presence or absence of RAPD marker UBC30, and average survival of flue-cured tobacco cultivars against races 0 and 1 of Phytophthora parasitica var. nicotianae.w
w Percent survival data for race 0 was obtained from nine field experiments conducted at seven locations from 1999 to 2004. Final survival of race 1 indicator TI 1071 averaged 96% compared to 44% for the standard susceptible cultivar K 326. Percent survival data for race 1 was obtained from nine field experiments at five locations conducted between 2000 and 2006. Percent survival of TI 1071 in the race 1 experiments averaged only 8% compared to 31% on K326.
x References 7 and 11 in literature cited.
y - = difference could not be calculated.
z nt = not tested;
Implications for Disease Management
Rotating the horizontal resistance to black shank derived from FL301 with the vertical resistance provided by the Ph gene has been recommended as the best strategy to minimize losses to tobacco black shank in fields containing races 0 and 1 of P. nicotianae (17). However, this strategy assumes that the source and nature of the black shank resistance in available cultivars is clearly known and that farmers can plant black shank resistant cultivars with or without the Ph gene. Our results indicate that the number of flue-cured tobacco cultivars currently available that could be appropriately used to shift P. nicotianae populations from mostly race 1 to predominantly race 0 is limited (with the exception of GL 330) to pure-bred cultivars released 10 or more years ago. Rotating most currently popular or available cultivars would not alleviate selection pressure favoring race 1 of the pathogen, because most of these possess the Ph gene.
Whether or not tobacco farmers adopt a cultivar-rotation strategy to manage black shank, reducing their disease losses in the short term will require additional effective use of soil fungicides. However, frequent use of these materials has been demonstrated to lead to reduced sensitivity by the pathogen (14). Consequently, increasing levels of partial resistance to both races of black shank and/or incorporating effective resistance to the disease from other sources may be critical to long-term reduction in tobacco crop losses to P. nicotianae.
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