1992) 363

BACTERIAL R I N G ROT DISEASE D E V E L O P M E N T IN RESISTANT AND SUSCEPTIBLE CULTIVARS

EE. Manzer 1 and C. J. Kurowski, Jr . 2

Abstract

In two greenhouse tests, we compared cell populations of Corynebac- terium sepedzmicum in inoculated cultivars which had failed to show symptoms in field inoculation tests against cultivars that produce well-defined symp- toms. Both population studies showed that cell numbers as high as 1 x 109 colony-forming units per grn of fresh weight (cfu gm -1) were reached in some cultivars prior to symptom development. In one of these tests, con- ducted when day length averaged 14�89 hrs, some cultivars failed to show symptoms 11 wks after inoculation but were found to have cell populations as high as 3.2 x 108 cfu gm -1 7 wks after inoculation. In another study, root and seed piece inoculations were compared in susceptible and resistant cul- tivars and only minor differences in symptom expression due to mode of entry of the pathogen were found.

Introduction

Diagnosis of bacterial ring rot (BRR) caused by C o r y n e b ~ m sepedom- cum (Spieck. & Kotth.) Skapt. & Burkh. [syn. Clavibacter michiganense subsp. sepedonicus (Spieck. & Kotth.) Davis, eta/. ] is, at least in large seed lots, still initially dependent upon symptom expression. Likewise, most of the searches for resistance to B R R have been based on symptom expression. Manzer and McKenzie (6) documented, over several seasons, the variable reaction to B R R infection of many named cultivars and seedling selections. They showed that both the number of days required for symptom expression and the intensity of expression are variable among cultivars. In particular, the cultivar BelRus consistently failed to show definitive symptoms of B R R in field inoculation trials in Maine (5, 6) although it has been found to sup- port high numbers of BRR bacteria (5 and Kurowski, unpublished).

Our present studies on resistance to BRR (3) were begun prior to any reports on actual populations of C. sepedonicum ceils in host tissue relative to symptom expression (1). Our main objective was to determine whether

1Professor and Research Associate respectively, Department of Plant Biology and Pathol- ogy, University of Maine, Orono, ME 04469. Tel: (207) 581-2985. Maine Agricultural Experiment Station Publication No. 1629. 2Present address: Harris Moran Seed Company, Research & Development, 100 Breen Road, San Juan Bautista, CA 95045. Accepted for publication November 30, 1991. ADDITIONAL KEY WORDS: Symptom expression, disease severity, Corynebacterium sepedonicum.

364 AMERICAN POTATO JOURNAL (Vol. 69

or not BRR reactions could be related to numbers of bacteria in the host and thereby differentiate host resistance to pathogen development from resis- tance to symptom expression only. In addition, since our BRR reaction studies in the field were all based on seed piece inoculation, a second objec- tive was to determine whether or not certain cultivars showing resistance to symptom expression in our field tests could react differently when root- inoculated.

Materials and Methods

Symptom Expression vs Cell Populations

In a preliminary experiment, BelRus was compared with WYl122 (USDA 47096 selfed), a selection obtained from Dr. W. A. Riedl in 1961, which had shown resistance to BRR symptom expression in several field inoculation trials. We also included: Katahdin, which develops symptoms relatively late; Norchip, which develops symptoms relatively early; and Mir- ton Pearl, which has shown extremely early symptom development. These latter three cultivars all have shown well-developed classical BRR symp- toms under Maine field conditions.

Total numbers of rooted stem cuttings of the five cultivars were as fol- lows: Katahdin, 36; Norchip, 67; BelRus, 72; WYl122, 67; and Mirton Pearl, 71. When these cuttings were 3-6 cm tall, their roots were dipped for 20 min in a 106 cfu/ml suspension of a streptomycin-resistant strain of C. sepedonicurn obtained from N.C. Gudmestad (NDSU 5-4). This isolate was chosen to facilitate culturing of macerated tissue samples to obtain popu- lation numbers and it had been shown to have disease-producing capacity equal to non-resistant isolates (Kurowski unpublished). All cultures used for inoculum were grown at 23 C in nutrient broth yeast-extract agar (NBY) for 2-3 days, then centrifuged and resuspended in quarter-strength nutri- ent broth. Immunofluorescent antibody staining (IFAS) of each sample was also performed, as described by DeBoer and McNaughton (2), to ensure that the population counts were those of C. sepedonicum. Conjugated antise- rum was obtained from Agdia, Inc., Elkhart, IN.

All plants were grown in 13 cm pots containing metro-mix 200 (W.R. Grace & Co., Cambridge, MA) in the greenhouse and watered as needed with tap water. Plants were given supplemental light using phosphor-coated (400 watt) MultiVapor Lamps (100 uE M -1 S -t) and fertilized first with a teaspoon ofosmocote granules (14-14-14), controlled release fertilizer, (Sierra Chemical Co., Mulipitas, CA) then every other wk with water soluble macro (15-15-15) and micronutrients (Peters Fertilizer Products, Fogelsville, PA).

Starting three wks after inoculation, 12 plants of each cultivar (6 of Katahdin) were randomly harvested at weekly intervals for five wks and placed in an ultrafreezer ( -73 C) until they were processed. Stems were

1992) MANZER AND KUROWSKI: BACTERIAL RING ROT 3 6 5

surface-sterilized for one minute in 0.525% sodium hypochlorite and rinsed three times in a 0.02 M sterile potassium phosphate buffer (KPB), at a pH of 7.2. A 0.5 g piece of stem was excised aseptically from the basal portion of the stem and ground in a sterilized mortar. All cell populations are reported as colony-forming units per gram fresh wt (cfu gm-1). Ten-fold serial dilutions, (10 -1 to 10-7), of each sample were made with 0.9 ml steri- lized KPB blanks. Replicate spread plates of each dilution were prepared by placing 100 ul of each dilution on NBY plates containing 100 ppm strep- tomycin. Dilutions from 10 -4 to 10 -7, were plated using a petri dish turn- table and a bent glass rod to spread the sample across the plate. All procedures were conducted under a laminar flow hood and each plate was individually wrapped with parafilm and placed in an incubator at 23 + / - 1 C. Plates were examined periodically for two wks to determine popula- tions of C. sepedonieum. Colony populations were determined using a dark field colony counter.

Disease severity ratings (4) were taken at each weekly harvest on all plants, including those being sampled. This study was conducted during a period when day length averaged slightly under 11 hrs. Average green- house temperatures ranged from about 17 C at night to 22 C during the day.

A second and more extensive study of symptom expression and cell populations was conducted involving 12 resistant and susceptible cultivars (Table 2). Methods used were as previously described except that melon- balled seed pieces were dipped in inoculum rather than inoculating roots as before. In addition, data were transformed to log10 for analysis. The dura- tion of this study covered a period (22 Mar. to 7 June, 1990) when day length under greenhouse conditions averaged approximately 14�89 hrs.

Root vs Seed Piece Inoculation

Three cultivars from the population study, WYl122, Norchip and Katahdin were chosen for this test. A total of 60 melon-balled seed pieces of each cultivar were cut and 30 of each were dipped for 5 rain in a 106 cfu/ml suspension of C. sepedonicum which was prepared as previously described. Both the inoculated and uninoculated seed pieces were planted on 23 March, 1989. Three weeks later, when all plants were 3-6 cm high, the previously uninoculated ones were removed from the pots. The roots were washed in tap water, dipped for 5 min in inoculum which had been prepared as previously described and then replanted. Disease severity rat- ings were made at 53, 65, 78 and 85 days after planting. No IFAS testing was done in this experiment which was conducted during a period when the average day length was approximately 14�89 hrs (23 Mar to 22 June, 1989). Greenhouse procedures and temperatures were similar to those of the previous experiment except that maximums during the day were occa- sionally 5 C higher in May and June.

366 AMEPdCAN POTATO JOURNAL (Vol. 69

Results and Discussion

Cell populations of C. sepedonicum in all cultivars in the preliminary experiment showed high numbers prior to symptom expression and paralleled development of BRR symptoms with time thereafter (Table 1). As expected (McKenzie and Manzer, unpublished), cell numbers and symp- tom expression developed rapidly in Mirton Pearl but, while symptoms continued to increase throughout the sampling period, cell numbers reached a peak at wk 5. A surprising result was that greatest symptom development occurred in BelRus. We suspect that this latter result may have been a manifestation of the short day length during which the experiment was con- ducted (7). Cell populations were lowest in WYl122 but, unlike its behavior in field trials, typical BRR symptoms were observed. This result, again, suggests that short days may have caused the increased development. Symp- toms continued to develop in the plants of each cultivar that remained at the end of the five-week sampling period. Throughout this experiment, IFAS tests confirmed that the cell count data were of C. sepedonicum.

The second population study agreed with the first in that high cell populations were more or less associated with degree of symptom expres- sion (Table 2). Likewise, appreciable, but not significantly different cell populations were detected in all cultivars 5 wks after inoculation but only Mirton Pearl showed any symptoms (2 of 6 plants each rated 1), therefore data at 5 wks are not reported. Population differences among weeks was nonsignificant but differences among cultivars and interaction of cultivars and weeks were highly significant (P =.01). This study also indicated that, while none of the entries was immune, 11 wks after inoculation several of them showed little or no symptom development. Of these, AF1332-52 had a final cell count of 4 x 10 s cfu gin -1, down from a high of 4.3 x 108 at 7 wks and B3478-23 slowly increased from 4.3 x 10 ~ cfu grn -1 at 5 wks to 1.9 x 105 at 11 wks. Though the cultivars are different from those tested by DeBoer and McCann (1), the range in numbers of cells they obtained by immunofluorescence counts are in general agreement with our culture plate counts.

Comparison of root and seed piece inoculations showed that, though roots were inoculated three weeks later than the seed pieces, expression of BRR symptoms began at about the same time for each (Table 3). Progres- sion of symptoms in root-inoculated Katahdins was ahead of that in Nor- chip though their positions were reversed in the previous experiment. In addition, symptoms progressed more slowly in all three root-inoculated cul- tivars in this experiment relative to the previous one. These results again suggest that day length may be an important factor. Of special note in this regard was the performance of WY1122 which produced only marginally discernible symptoms following each inoculation method under the longer days more comparable to field conditions. Analysis of variance showed that

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1992) MANZER AND KUROWSKI: BACTERIAL RING ROT 369

TABLE 3.--Average disease severity values comparing seed piece and root inoculation of susceptible (S) and resistant (R) adtivars,

Days after Planting 1 Cultivar 53 65 78 85 Norchip (S)

Seed 0.67 1.33 2.47 3.40 Root 0.10 0.60 1.63 2.43

Katahdin (S) Seed 0.60 1.07 1.47 2.47 Root 0.40 0.97 1.97 3.40

WYl122 (R) Seed 0.14 0.14 0.28 0.28 Root 0.10 0.17 0.13 0.33

Analysis of Variance F-Ratios 2

Source Seed vs Root 7.96" * 2.39 0.66 0.10 Cultivar 3 6.89" * 19.73 * * 66.15" * 80.62 * * Interaction 2.82 3.71 * 4.22" 3.54" Error (Mean sq.) 0.222 0.339 0.321 0.411 1Sixty seed pieces of each cultivar, 30 of which were inoculated, were planted on 23 Mar. The remaining 30 plants were root-inoculated 20 days later. A suspension of 10%fu/ml of C. sepeabni cum was used for each inoculation. 2*=significant at P=0.05, **=significant at P=0.01 3Katahdin and Norchip were not significandy different at P=0.05.

differences in ratings due to inoculat ion me thod were significant (P--.01) only when symptoms first began to appear. In early readings, s y m p t o m deve lopment progressed m o r e rapidly in seed-inoculations whereas later readings showed a small bu t statistically significant (P =.05) reversal. As expected, the disease react ion of WY1122 was significantly lower (P = 0.01) than ei ther Ka tahd in or Norchip.

While direct comparisons of root and seed piece inoculations were not repeated, results of seed piece inoculations in m a n y field tests show symp- tom development similar to results obta ined in the popula t ion studies repor ted here. Therefore , we conclude that, in the cultivars we have com- pared, wide differences in disease reaction app ea r not to be due to m o d e of entry of the pathogen. All results reported here do, however, suggest that, as in our field trials, symptom-deve lopment under long day conditions can be appreciably a t tenuated relative to short day conditions.

We did not find a clear basis for differentiating resistance to the B R R pa thogen and resistance to s y m p t o m expression in the popula t ion studies. While we did show that such selections as WYl122 and B3478-23 were con-

370 AMERICAN POTATO JOURNAL (Vol. 69

sistently a m o n g the lowest in cell populat ions, there was significant varia- tion dependen t upon when the sampl ing was done. W h e t h e r or not lower populat ions of C. sepedonicum ceils have less effect on p lant growth and product iv i ty is not known at present. As DeBoer and M c C a n n (1) state, disease ratings are somewhat variable depending upon growing conditions and inoculum concentrat ion. All work on resistance to B R R mus t there- fore take these factors into account and appropriate serological testing must also be included to avoid the danger of releasing symtomless carriers into the industry.

Acknowledgments

This work was supported in part by U S D A Gran t NO. 58-3244-8-040. T h e authors wish to thank Dr. Nell C. Gudmes tad , Nor th Dakota State Univers i ty for use of the s t reptomycin-resis tant m u t a n t of C. seped~nicum ( N D S U 5-4). Special thanks is extended to Dr. David H. L a m b e r t for statistical help and for extensive editorial review.

Literature Cited

1. DeBoer, S.H. and M. McCann. 1990. Detection of Corynebacterium sepedonicum in potato cultivars with different propensities to express ring rot symptoms. Am Potato J 67:685-694.

2. DeBoer, S.H. and M.E. McNaughton. 1986. Evaluation ofimmunofluorescence with monoclonal antibodies for detecting latent bacterial ring infections. Am PotatoJ 63:533-543.

3. Kurowski, C.J. and EE. Manzer. 1991. Reevaluation of Solanum species accessions show- ing resistance to bacterial ring rot. Am Potato J 69:289-298.

4. Logsdon, Charles E. 1967. Effect of soil temperature on potato ring rot. Am Potato J 44:281-286.

5. Manzer, Franklin. 1983. Reaction of the BelRus variety to bacterial ring rot infec- tion - an update. Maine Agricultural Experiment Station. Misc. Report No. 283, 4 pp.

6. Manzer, EE. and A.R. McKenzie. 1988. Cultivar response to bacterial ring rot infec- tion in Maine. Am Potato J 65:333-339.

7. Nelson, G.A. and G.C. Kozub. 1983. Effect of total light energy on symptoms and growth of ring rot-infected Red Pontiac potato plants. Am Potato J 60:461-468.