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© 2008 Plant Management Network.
Accepted for publication 27 February 2008. Published 18 June 2008.


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


Corresponding author: Charles S. Johnson. spcdis@vt.edu


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.


Abstract

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.


Introduction

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).


 

Fig. 1. Chlorotic and dying plants in a commercial flue-cured tobacco field infested by race 1 of Phytophthora nicotianae in Pittsylvania Co., VA in 2005.

 

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.

Entry no. Cultivar Year released Pedigree
9 K 399 1979 (C 139 × C 319) × NC 95
49 K 326 1981 McNair 225(McNair 30 × NC 95)
30 K 358 1987 McNair 926 × 80241
42 K 346 1988 McNair 926 × 80241
44 GL 939 1992 McNair 926 × 80241
10 K 149 1988 ([G-28×354]×[CB 139×F 105] ×
[G-28×354])McNair 399
15 RG 17 1993 K 326 × K 399
3 SP NF3 1996 Speight NF 1 × NC 0007
14 NC 606 1998 NC 729 × NC 82
36 SP 210 2000 (SP 116 × G-126)(K 346 × SP G-28)
4,41 GL 330 2005 McNair 926 × 80241
51 Coker 371-Gold 1986 (G-28×354)×(CB139×F105)
(G-28×34)×NC82
50 NC 71 1995 Hybrid
18 SP 168 1996 Coker 371-Gold × SP G118
48 NC 72 1996 Hybrid
38 SP 179 1997 Coker 371-Gold × SP G-28
23 NC 291 1997 Hybrid
29 NC 297 1998 Hybrid
43 RG H51 1998 Hybrid
11 SP H20 1999 Hybrid
8 NC 810 2000 OX 2102 × NC 729
46 NC 102 2001 Hybrid
47 NC 299 2001 Hybrid
21 SP 220 2002 (K 346 × SP 117)(SP 116 × K 346)
7 NC 196 2002 Hybrid
2 GL 350 2003 Hybrid
24 CC 27 2003 Hybrid
28 NC 471 2003 Hybrid
33 SP 225 2003 (SP 168 × K 346)(SPA 95 × SP 168)
5 SP 227 2003 (SP 151 × K 346)(SP 202 × K 346)
45 SP 234 2004 (SP 168 × K 346)

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).


 
A

B
 
 

Fig. 2. Coupling-phase RAPD marker UBC30 expressing polymorphism between resistant and susceptible cultivars of tobacco to Phytophthora nicotianae. The resistance marker has a product band size of 490 bp.
(A) The first 26 cultivars: L = molecular size marker, 1 = CU195, 2 = GL350, 3 = SPNF3, 4 = GL330, 5 = SP227, 6 = ULT135, 7 = NC196, 8 = NC810, 9 = K399, 10 = K149, 11 = SPH20, 12 = XCC1, 13 = NCTG121, 14 = NC606, 15 = RG17, 16 = NC5, 17 = NC6, 18 = SP168, 19 = RGH4, 20 = XP2035, 21 = SP220, 22 = NC7, 23 = NC291, 24 = CC27, 25 = SP236, 26 = RX123.
(B) The last 26 varieties: 27 = XP2110, 28 = NC471, 29 = NC297, 30 = K358, 31 = KT204, 32 = NCTG135, 33 = SP225, 34 = RX121, 35 = SP235, 36 = SP210, 37 = CC700, 38 = SP179, 39 = KTH2405, 40 = RJR13, 41 = GL330, 42 = K346, 43 = RGH51, 44 = GL939, 45 = SP234, 46 = NC102, 47 = NC299, 48 = NC72, 49 = K326, 50 = NC71, 51 = C371Gold, C = negative control.

 

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

Entry no. Cultivar Ph gene
reported
x
Presence
of marker
% survival
against:
Differ-
ence
y
Race 0x Race 1
9 K 399 66 67 1    
49 K 326 44 31 13    
30 K 358 53 32 21    
42 K 346 79 69 10    
44 GL 939 52 50 2    
10 K 149 57 54 3    
15 RG 17 56 29 27    
3 SP NF3 63 60 3    
14 NC 606 64 57 7    
36 SP 210 73 51 22    
4,41 GL 330  ntz nt -
51 Coker 371-Gold + + 97 49 48    
50 NC 71 + + 97 51 46    
18 SP 168 + + 99 75 24    
48 NC 72 + + 97 43 54    
38 SP 179 + + 99 59 40    
23 NC 291 + + 99 49 50    
29 NC 297 + + 95 44 51    
43 RG H51 + + 92 43 49    
11 SP H20 + + 98 56 42    
8 NC 810 + 85 63 22    
46 NC 102 + + 97 29 68    
47 NC 299 + + 97 39 58    
21 SP 220 + 99 64 35    
7 NC 196 + 97 53 44    
2 GL 350 + 94 57 37    
24 CC 27 + 99 35 64    
28 NC 471 + + 99 65 34    
33 SP 225 + 97 87 10    
5 SP 227 + 99 69 30    
45 SP 234 + + nt 53 -

 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.


Literature Cited

1. Apple, J. L. 1962. Physiological specialization within Phytophthora parasitica var. nicotianae. Phytopathology 52:351-354.

2. Apple, J. L. 1967. Occurrence of race 1 of Phytophthora parasitica var. nicotianae in North Carolina and its implications in breeding for disease resistance. Tobacco Sci. 11:79-83.

3. Carlson, S. R., Wolff, M. F., Shew, H. D., and Wernsman, E. A. 1997. Inheritance of resistance to race 0 of Phytophthora parasitica var. nicotianae from the flue-cured tobacco cultivar Coker 371-Gold. Plant Dis. 81:1269-1274.

4. Csinos, A. S. 2005. Relationship of isolate origin to pathogenicity of race 0 and 1 of Phytophthora parasitica var. nicotianae on tobacco cultivars. Plant Dis. 89:332-337.

5. Csinos, A. S., and Bertrand, P. F. 1994. Distribution of Phytophthora parasitica var. nicotianae races and their sensitivity to metalaxyl in Georgia. Plant Dis. 78:471-474.

6. Davis, D. L., and Nielsen, M. T. 1999. Tobacco: Production, Chemistry and Technology. Blackwell Publ., Boston, MA.

7. Johnson, C. S. 2006. Disease control. Pages 37-50 in: 2007 Flue-Cured Tobacco Production Guide. T. D. Reed, ed. Virginia Coop. Ext., Blacksburg, VA.

8. Johnson, E. S., Wolff, M. F., and Wernsman, E. A. 2002. Marker-assisted selection for resistance to black shank disease in tobacco. Plant Dis. 86:1303-1309.

9. Johnson, E. S., Wolff, M. F., Wernsman, E. A., Atchley, W. R., and Shew, H. D. 2002. Origin of the black shank resistance gene, Ph, in tobacco cultivar Coker 371-Gold. Plant Dis. 86:1080-1084.

10. Litton, C. C., Stokes, G. W., and Smiley, J. H. 1966. Occurrence of race 1 of Phytophthora parasitica var. nicotianae. Tobacco Sci. 10:73-74.

11. Mila, A., and Broadwell, A. 2006. Managing disease. Pages 158-186 in: Flue-Cured Tobacco Guide, 2007. Agric. Ext. Serv. Publ. No. AG-187 (rev)., Dept. of Plant Pathol., North Carolina State Univ., Raleigh, NC.

12. Mila, A., Gutierrez, W., and Broadwell, A. 2006. Extension-research flue-cured and burley tobacco pathology program annual report. Dept. of Plant Pathol., North Carolina State Univ., Raleigh, NC.

13. Powell, N. T., Porter, D., Wood, K., Conniff, L., and Wickham, P. 1987. Extension-research tobacco pathology program summary report. Agric. Ext. Serv. Publ. No. AG-191 (rev)., Dept. of Plant Pathol., North Carolina State Univ., Raleigh, NC.

14. Shew, H. D. 1985. Response of Phytophthora parasitica var. nicotianae to metalaxyl exposure. Plant Dis. 69:559-562.

15. Shew, H. D., and Lucas, G. B. 1991. Compendium of Tobacco Diseases. American Phytopathological Society, St. Paul, MN.

16. Stokes, G. W., and Litton, C. C. 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.

17. Sullivan, M. J., Melton, T. A., and Shew, H. D. 2005. Managing the race structure of Phytophthora parasitica var. nicotianae with cultivar rotation. Plant Dis. 89:1285-1294.

18. Valleau, W. D., Stokes, G. W., and Johnson, E. M. 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.