lated 'Red Pontiac' plants were grown under low light intensity, and .... symptoms and growth of ring rot-infected Red Pontiac potato plants. ... plants in Montana.
Euphytica 82: 125-132,1995.
© 1995 Kluwer Academic Publishers. Printed in the Netherlands.
125
Immunity to Clavibacter michiganensis subsp. sepedonicus: Screening of exotic Solanum species C.l Kriel 1 , S.H. Jansky2, N.C. Gudmestad3 & D.H. Ronis 4
I
1 Department ofHorticulture and Forestry, North Dakota State University, Fargo, ND 58105, USA; 2 Department ofBiology, University of Wisconsin-Stevens Point, Stevens Point, WI54481, USA; 3 Department of Plant Pathology, North Dakota State University, Fargo, ND 58105, USA; 4 McCain Produce, Inc., P.O. Box 68, Florenceville, NB, Canada, EO] 1KO
Received 26 July 1994; accepted 9 January 1995
Key words: bacterial ring rot, disease screening, immunity, potato, Solanum tuberosum, Clavibacter michiganensis, sepedonicus
Summary
Accessions from exotic Solanum species, including diploid and tetraploid species, were screened for immunity to Clavibacter michiganensis subsp. sepedonicus, the causal agent of potato ring rot. The diploid species included S. infundibuliforme, S. lesteri, S. megistacrolobum, S. tuberosum Group Phureja, S. polyadenium, S. pinnatisectum, S. raphanifolium, S. sparsipilum, S. sanctae-rosae, S. tuberosum Group Stenotomum, S. toralapanum, and S. verrucosum. The tetraploid species included S. tuberosum Group Andigena, S. acaule, S. fendleri, S. hjertingii, S. oplocense, S. polytrichon, and S. stoloniferum. Apparent immunity was initially found in several diploid species, but was not present during subsequent retesting. Immunity was found in nine accessions of tetraploid S. acaule. These accessions maintained their immunity during testing over an eight-month period. S. acaule appears to be a good source of immunity for introgression studies.
er & McKenzie, 1988). De Boer & McCann (1990) found high population levels of C. m. subsp. sepe Bacterial ring rot of potato (Solanum tuberosum), donicus in all tuber samples from cultivars that pro caused by Clavibacter michiganensis subsp. sepe duced only 9% symptomatic tubers. Resistant cultivars donicus (Spieck & Kotth.) Davis et aI., is a potentially have been developed but have been found to harbor devastating disease in the potato industry. Manage high populations of C. m. subsp. sepedonicus without ment of this disease is accomplished primarily through the expression ofsymptoms (Bonde et aI., 1942). These the use of certified seed (Gutbrod, 1987; Racicot et aI., types of cultivars have been termed symptomless carri 1938). Field detection of ring rot is often difficult due to ers. The association of resistance with the potential of the lack of typical symptom development, which can symptomless infections has prevented many cultivars be affected by environment (Bishop & Slack, 1981; with ring rot resistance from becoming commercially Bishop & Slack, 1987; Logsdon, 1967; Nelson & accepted (Bonde et al., 1942; Manzer & McKenzie, Kozub, 1983), inoculum dose (Bishop & Slack, 1987; 1988). Nelson, 1982), cultivar response (DeBoer & McCann, Immunity to a pathogen means that the host is not 1990; Manzer & Mckenzie, 1988), and other pathogen capable of being infected. The purpose of this study infections (Nelson & Kozub, 1987; Sun & Easton, was to screen wild Solanum species for immunity to 1983). C. m. subsp. sepedonicus. High bacterial population levels have been found in
both tuber and vine samples from cultivars that do not
express symptoms (De Boer & McCann, 1990; Manz Introduction
-
126 Materials and methods Initial screening
True potato seed from 26 accessions of 14 different wild and semi-cultivated Solanum species that had been noted to exhibit resistance to ring rot was obtained from the IR-1 Potato Introduction Station in Sturgeon Bay, Wisconsin (Hanneman & Bamberg, 1986). Plant introduction numbers of accessions were as follows: S. acaule 175396,210029,472638,472640,472643, 472655, 472651, 473323, and 473327; S. tubero sum Group Andigena 214421 x 214431,214435, and 225637; S. infundibuliforme474522; S. lesteri 442694; S. megistacrolobum 458348, 473130, and 473140; S. oplocense 473187; S. tuberosum Group Phure ja 195191; S. polyadenium 347768; S. pinnatisec tum 347766; S. raphanifolium458384; S. sparsipilum 310959; S. tuberosum Group Stenotomum 458393; S. toralapanum 458396; S. verrucosum 161173. Approximately 50 seeds of each accession were germinated in Jiffy mix T M, and kept moist on a cap illary mat. Seedlings were transplanted at the four-leaf stage into Sunshine mix T M in 15.2 cm diameter sterile pots and grown in a greenhouse under 18-hour days with high intensity sodium lights. They were fertilized biweekly with 100 ppm Peters water-soluble 20-20 20. Roots and basal stems of four-week-old plants were inoculated with C. m. subsp. sepedonicus strain NDCs OFF. All inoculations were performed as described below. Symptoms were noted at six weeks after inoc ulation. Symptoms observed, typical of C. m. subsp. sepedonicus infections, included interveinal chlorosis, marginal leaf necrosis, and wilting. Stem tissue sam ples were prepared for indirect immunofluorescence antibody staining (IFAS) to determine populations of C. m. subsp. sepedonicus (De Boer & Wieczorek, 1984; De Boer & McNaughton, 1986; Gudmestad et aI., 1991). Second screening
Five to 50 seedlings from each of 14 accessions, selected on the basis of initial screening results, and five other species were obtained from the IR-1 Potato Introduction Station. Plant introduction num bers were as follows: S. acaule 175396, 210029, 472638, 472640, 472643, 472651, 472655, 473323, and 473327; S. tuberosum Group Andigena 214435; S. infundibuliforme 473522; S. lesteri 442694; S. megistacrolobum 473140; S. sanctae-rosae 230464;
S. tuberosum Group Stenotomum 458393; S. fendleri 275156; S. hjertingii 251065; S. polytrichon 283106; S. stoloniferum 161170. Seeds were soaked in 1500 ppm GA3 for 24 hours to break dormancy (Spicer, 1961). Seedlings were han dled as described for the first screening and maintained as mother clones. For clonal maintenance, shoot tips were excised from the mother plants, placed in Sun shine mixT M, given a mist treatment for approximately four to six days, and brought into the main greenhouse for four to six weeks, until properly rooted. Then, cuttings were inoculated using a modified Nelson & Harper (1973) technique, as described below. Because symptomology of ring rot expression was observed to be atypical in many wild species during the first screening, efforts during the second screen ing concentrated on IFAS testing. Each mother clone was screened once, with one cutting per plant. Plant samples that resulted in a negative IFAS, indicating the presence of no C. m. subsp. sepedonicus bacteria, were designated as immune test clones. Replicated cuttings taken from mother clones were maintained as immune mother test clones for retesting. Stability of immune reactions to C. m. subsp. sepedonicus in immune test clones was investigated by repeated testing of plant material. Retesting was accomplished by rooting and inoculating five to ten stem cuttings, taken from each immune mother test clone, with C. m. subsp. sepedonicus at each trial peri od. The diploid immune test clones were retested when the immune mother test clones were eight (eight-month trial), 15 (I5-month trial) and 19 months old (I9 month trial). Tetraploid S. acaule species were divided into two evaluations. Twelve tetraploid immune test clones were maintained in the greenhouse and retest ed when the mother clones were eight (eight-month trial), 15 (I5-month trial) and 19 months (19-month trial). Twenty-four tetraploid immune test clones were regrown from tubers and retested when the mother clones were four (four-month trial) and six months old (six-month trial). Plant inoculations
All inoculations were carried out with NDCs-OFF strain of C. m. subsp. sepedonicus obtained from the NDSU Plant Pathology Department. Aliquots kept at - 80° C were grown on nutrient broth yeast extract (NBY) agar (Schaad, 1980). Bacterial cultures were incubated at 23° C for three to five days. Verification of cultural purity was by colony morphology and a
127 Gram stain. The plates were harvested by washing with 0.05 M phosphate buffer, pH 7.2. The bacterial suspen sions were photometrically adjusted to an OD64o = 0.1, which is approximately 108 colony forming units (cfu) per ml, as established on a standard curve for NDCs OFF. A 106 cfu/ml inoculum was prepared by making two ten-fold dilutions (Bishop & Slack, 1987), using one-quarter strength NBY to maintain cell viability (Nelson, 1982). For uniform and efficient disease transmission, root inoculation was performed by washing the plant roots clean of soil media and placing them in 106 cfu/ml inoculum for one to three hours, depending on the light conditions (Nelson et aI., 1971; Sherf, 1949). Successful infection and bacterial uptake is more rapid under high light intensity than low light intensity. The cultivar Red Norland was used for both positive (c. m. subsp. sepedonicus inoculated) and negative (one quarter strength NBY inoculated) controls. The plants were placed in 10.2 cm diameter plastic pots filled with Sunshine mixT M and arranged in a completely random design in growth chambers (Bishop & Slack, 1987). For optimum growth, plants were kept at 23° C days and 18° C nights and grown under a 12-hour photope riod (Logsdon, 1967). Irradiance was measured by a Lamda LI-185 Quantummeter, with a total light energy of 45 mW cm- 2 hr- 1 (Nelson & Kozub, 1983). Plants were fertilized with four grams of 14-14-14 Osmo cote (Sierra Chemicals, Mulpitas, Cal.) one week after inoculation. Populations oiC. m. subsp. sepedonicus
Populations of C. m. subsp. sepedonicus were deter mined by indirect immunofluorescence antibody stain ing (IFAS) (De Boer & Wieczorek, 1984; De Boer & McNaughton, 1986). A single sample of stem tis sue (0.5 g) was aseptically removed and macerated in 1 ml 0.01 M phosphate buffered saline (PBS), pH 7.2, and processed as previously described (Gudmestad et aI., 1991). Undiluted supernatant and three tenfold dilutions made in 0.01 M PBS were pipetted onto multiwelled toxoplasmosis slides (Belleo Glass, Inc., Vineland, N.J.) to which antibody and conjugate was added. Positive controls consisted of four tenfold dilu tions of C. m. subsp. sepedonicus placed on multi welled slides. The negative control slide was 0.01 M PBS. Mouse anti-Cs monoclonal 9Al serum and fluo rescein isothiocynate conjugated goat anti-mouse IgC and IgM were purchased from Agdia Inc., Elkhart, IN.
Antibody concentrate and conjugate were consecutive ly diluted in 0.01 M PBS to the appropriate strength and applied to each slide well. Slides were incubated at 37° C for one hour, rinsed with distilled water, and air dried. Mounting fluid (10: 1 glycerol:PBS) and a cover slip were applied and the slides were viewed at 1250 x on an Olympus BH-2 photomicroscope equipped with a fluorescent illuminator and light source. C. m. sub sp. sepedonicus cells were identified as those with bright green fluorescence and smooth, sharply outlined curved, rods 0.3-0.5 x 0.6-0.9 p,m in size. The first two dilution wells, containing the undiluted extract and the 10- 1 dilution, were exhaustively searched; otherwise, ten random microscope fields per well were viewed. Cells were counted in the well with the optimal con centration. The IFAS procedure described above was modified to increase sensitivity and the reliability of detecting low populations of C. m. subsp. sepedonicus. Plant extract was placed in 1.5 ml microcentrifuge tubes and vortexed on a Vortex-2 Genie (Scientific Indus tries, Bohemia, N.Y.). Plant debris was pelleted by centrifuging the extract at 5000 g for 1 minute in a Bax ter Biofuge A Centrifuge and the pellet was discard ed. Three tenfold dilutions were made from the super natant with a two-minute vortexing between dilutions. The diluted samples were prepared for immunofluo rescent antibody staining and, if they gave a negative IFAS, the supernatant was re-centrifuged at 12,000 g for 15 minutes. The resulting pellet was resuspended in 40 p,l PBS, and three ten-fold dilutions prepared for immunofluorescence staining (Miller, 1984). Random checks were made on resuspended plant debris and discarded supernatant for cell loss or escape during the procedure. Analysis
Bacterial populations in the initial screening are expressed as immunofluorescing units/microscope field (IFUIMF) since they were based on randomly selected microscope fields. In subsequent trials, the entire microscope well, representing a specific dilu tion, was used. In these later trials, bacterial pop ulations, expressed as IFU/g, were determined with the formula P = MF*B*G (I), where P = popula tion, MF = l/(ml/field), B = immunofluorescing units (IFU)/highpowered field (1250 p,), and G = ml/g (Baer & Gudmestad, 1991). Analysis of variance of IFU/g for greenhouse-maintained clones and clones grown
128 Table 1. Bacterial ring rot symptoms and populations of C. m. subsp. sepedonicus during initial Solanum species screening
Results Initial screening
Species S. acaule
PI
Number l
175396 210029 472638 472640 472643 472655 472651 473323 473327 Group Andigena 214421 x 214431 214435 225637 S. infundibulijorme 473522 S. lesteri 442694 S. megistacrolobum 458348 473130 473140 S. oplocense 473187 S.phureja 195191 S. polyadenium 347768 S. pinnatisectum 347766 S. raphanijolium 458384 S. sparsipilum 310959 Group Stenotomum 458393 S. (oralapanum 458396 S. verrucosum 161173
Symptoms 2
lFUIMF3
LLD, UIMLN LLD LLD, UIMLN LLD NONE LLD LLD, UIMLN NONE
0.63 0.00 0.65 1,404 0.00 0.00 0.00 0.00
LLD
0.00
MLN
10.10
MLN LLD 27/36 5 MLN, LLD IVe, MLN LLD MLN 4/13 IVe, MLN IVe, MLN
LLD IVe, MLN, WLT 22/25 WLT, lVC MLN IVe, WLT
0.00 1.88 0.00 0.00 68.14 15.00 66.67 >100.00 52.47 >100.00 >100.00 57.50 54.15 1.384
NONE Ive >100.00 8/12 IVe, MLN, WLT 50.00
Plant introduction number.
LLD = Lower Leaf Death; U = Upper; MLN = Marginal Leaf
Necrosis; IVC =lnterveinal Chlorosis; WLT = Wilt.
3 Mean number of immunofluorescing units per microscope field
viewed at 1250 x (based on 2-13 randomly selected plants).
4 Both negative and positive IFAS results in plant samples.
5 Number of plants expressing symptoms/number of plants tested.
I
2
from tubers was performed using the Statistical Anal ysis System (Raleigh N.C.). In the initial trial, bacterial populations found in the plant samples were classified as immune (0 IFU/MF), resistant (1-10 IFUIMF), low resistant (11 20 IFU/MF) or susceptible (> 20 IFU/MF). In subse quent trials, populations of C. m. subsp. sepedonicus found in plant samples were classified as immune (0 IFU/g), resistant (10 1-102 IFU/g), low resistant (103_ 104 IFU/g) or susceptible (> 105 IFU/g).
Accessions 472643 and 473323 of S. acaule and S. tuberosum Group Stenotomum did not express symp toms of bacterial ring rot (Table 1). Only lower leaf death (LLD) was noted in seven accessions: S. acaule 210029,472640,472655, and 473327, S. tuberosum Group Andigena 225637, S. megistacrolobum 458348, and S. tuberosum Group Phureja 195191. Chlorosis and wilting were evident in four species: S. polyadenium, S. pinnatisectum, S. sparsipilum, and S. verrucosum. Eleven accessions had plant samples that resulted in a negative IFAS test (Table 1). The nine accessions with no apparent populations of C. m. subsp. sepedonicus during testing included S. acaule accession numbers 210029, 472643, 472655, 472651, 473323, 473327; S. tuberosum Group Andigena 214435; S. infundibuli forme 474522; and S. lesteri; 442694. Two accessions, S. acaule 472640 and Group Stenotomum 458393 were initially negative but C. m. subsp. sepedonicus popula tions were detected in subsequent testing. Second screening
Ring rot symptoms, such as wilting, interveinal leaf chlorosis, and upward leaf curl, were noted in Group Andigena, S. infundibuliforme, S. megistacrolobum, S. polytrichon, and S. stoloniferum (results not shown). In Group Andigena, 32% of the susceptible/low resis tant plants expressed wilting and appeared susceptible to C. m. subsp. sepedonicus. In S. infundibuliforme, 72% of the susceptible/low resistant plants expressed wilting, chlorosis, and leaf curl; one plant was stunted. One third of the S. megistocrolobum susceptible/low resistant plants exhibited upward leaf curl. One suscep tiblellow resistant S. polytrichon plant wilted, and one was stunted; one susceptible/low resistant S. stolonifer um plant was also stunted. None of the S. lesteri. S. sanctae-rosae, Group Stenotomum, S. hjertingii, or S. acaule plants expressed ring rot symptoms. The tetraploid 4EBN (Endosperm Balance Number, John ston et aI., 1980) Group Andigena did not have any plants that gave an immune response in the second screening. Among the diploid species, S. infundibuliforme. S. lesteri, S. megistacrolobum, and S. sanctae-rosae all had one plant with an immune response based on IFAS testing (Table 2). Two plants in Group Stenotomum also gave an immune response.
129 Table 2. Populations of C. m. subsp. sepedonicus during second screening of Solanum species PI Number l Number
Species
S. acaule
I
Screened
Class2 R
US
175396
22
103
12
0
210029
8
2
I
5
472638 472640
11 9
9 4
2 5
0 0
472643
21
7
7
472651
27
4 4
12 3
7 II
2 9 0
5 4 8
3 31 4
I I 1
10 4 10
53 4 5 64
I 2
7 3
3 5
0
0
0
0 3
24
0
24
472655
8
473323
11
Group Andigena
473327 214435
16 39
S infundibuliforme S lesteri S. megistacrolobum
473522 442694 473140
64
S sanctae-rosae Group Stenotomum Sfendleri
230464 458393 275156
11 10 33 5
S. hjertingii S. polytrichon S. stoloni/erum
251065
76
283106
175
0 2
161170
23 5
0
10 17
I 4
2
Diploid species immune test clones retested
Diploid immune test clones were re-evaluated in 8-, 15-, and 19-month trials. In the 8-month trial, S. infundibuliforme displayed wilting and leaf curl, S. megistacrolobum chlorosis, and S. sanctae-rosae wilt ing. The S. lesteri immune test clones died in the growth chamber, and Group Stenotomum immune test clones did not express symptoms. S. infundibuliforme test clones responded with 90.0, 70.0, and 10.0 IFU/MF in the 8-, 15-, and 19-month trials, respectively. S. tuberosum Group Stenotomum responded with 26.0 and 55.0 IFUIMF in the 8- and 15 month trials, and was not retested in the 19-month trial. All of the S. lesteri plants died after inoculation in the 8-month trial and responded with 10.0 lFUIMF in the IS-month trial. S. sanctae-rosae responded with 53.0 lFUIMF in the 8-month trial, was not retested in the 15 month trial, but gave an immune response when retest ed in the 19-month trial. S. megistacrolobum responded with low bacterial populations of 0.3, 6.3, and < 0.1 IFUIMF, respectively, in the three trials. S. acaule accessions
Plant introduction number.
2 Classes of immunofluorescing units per microscope field,
1250 x, undiluted sample:
Immune (I) 0 lFUIMF; Resistant (R) < 1-10 lFUIMF;
Low resistant/susceptible (US) = > 11 lFUIME
3 Number of plants found in this class.
4 Plants expressed symptoms in this class.
5 Many plants died in growth chamber; they were assumed to be
susceptible.
6 Cuttings did not root for inoculation; five died during inoculation.
=
=
Five 4 x 2EBN species were screened for stem populations of C. m. subsp. sepedonicus after inoc ulation. No immune plants were found in S. fendleri, S. hjertingii, or S. stoloniferum accessions (Table 2). Two S. polytrichon plants gave an immune response but were not subjected to further testing because a decision was made to concentrate on S. acaule clones. Test clones with potential immunity were identified in all nine accessions of S. acaule based on IFAS testing of basal stem tissue (Table 2). S. acaule had extensive lower leaf death, but this is typical of this species when grown in the greenhouse. A total of 51 plants (38.3%) had an apparent immune response giving a negative IFAS result for C. m. subsp. sepedonicus. These acces sions were further evaluated for immunity to ring rot and are referred to as immune test clones.
Thirty-five S. acaule immune test clones were retest ed at eight months. Five to 10 replications of each of the 12 immune test clones 210029.9, 472643.1, 472643.9, 472643.10, 472643.23, 472651.22, 472655.4,472655.8,473327.5,473327.7,473327.15, and 473327.17 gave an immune response based on symptomology. Nine test clones, 210029.7,472640.4, 472643.2,473323.1, 473323.7,473327.4,473327.6, 473327.8, and 473327.13, had replications that result ed in both positive and negative IFAS tests. The twelve apparently immune test clones contin ued to be maintained in the greenhouse and were evalu ated again when the mother clones were 15 (IS-month trial) and 19 months old (l9-month trial) (Table3). Sus ceptible control clones, 472640.1 and 473323.5, had C. m. subsp. sepedonicus populations of 5.5 x 105 and 1.3 x 107 IFU/g, respectively, at the IS-month trial. Test clones 472643.19, 473323.1, and 473327.10 also gave a susceptible response with bacterial populations> 106 IFU/g. Bacterial population levels of less than 4.2 x 104 IFU/g, indicating low resistance, occurred in test clones 210029.9, 472643.1, 472651.22, 472651.28, 472655.4,472655.8,473323.7, and473327.7. Of these clones, 210029.9, 472651.22, 472655.8, 473327.1, and 473327.7 gave an immune response in the 19 month evaluation. C. m. subsp. sepedonicus popula
130 1iJble 3. Populations of C. m. subsp. sepedonicus in S. acaule immune test clones maintained in the greenhouse for 15 and 19 months PI Clonc l
15 Months lFU/g2
19 Months lFU/g
prr
PI.Clone l
prr a3
Oil 2
o
472640.5 6/7 472643.1 7/10 472643.19 2/5
1.6 x 105 a 9.3 x to' a l.l X 106 b
4/14
0.3 X 10' 2.0 X to-I
175396.8 175396.18
5.0 x 10- 1
472651.22 472651.28 472655.4 472655.8
2/8 5/5 2/5 2/6
OJ
10 1 a 4.2 x 104 a 1.2xI02 a 0.2 X 102 a
Oil 4
o
2/10
10 1 0.8 X tO l
175396.21 175396.22 175396.29
473323.1 473323.7 473327.7
5/5 3/7 1/6
5.7
2.1 X 102 a 0.2 X 10' a
473327.10 7/8 472640.1 4 3/6
l.5x106 b 5.5 X 105 b
473323.5 4
1.3
210029.9
I
Table 4. Populations of C. m. subsp. sepedonicus in S. acaule immune test clones grown from tubers and retested at two eval uation periods
6/6
5/5
3.0
X t0 2
X
x
X
106 bc
t07 C
1122 1110
0.7
6110 0/12
o
0/10
o
X
4114
0.2 X 10'
0/12
o
1116
3.0
X
3/12 3110
OJ
X
0.2
X 101
210029.7 472638.17 47263819 47263822 472640.4 472640.8
10- 1 10'
472643.2 472643.9 472643.10
Plant introduction number.clone number.
472643.23 472651.9 472651.14
2 Mean immunofluorescing units per g tissue.
3 Means that share the same letter do not differ significantly
(P =0.05), LS means.
4 Control (preselected susceptible clones).
tions in the two susceptible control clones, 472640.1 and 473323.5, were greatly reduced in the 19-month evaluation. Twenty-four S. acaule immune test clones, regrown from tubers, were tested in replication when mother clones were four and six months old (Table 4). Five clones, 473327.4, 473327.5, 473327.8, 473327.15, and 473327.17, showed a significant decrease in lFU per g between the four- and six-month trials. All clones had populations of less than 0.1 x 101 IFU/g. Six clones, 210029.7,472638.17,472638.19,472638.22, 472640.4, and 472651.14, had no detectable C. m. sub sp. sepedonicus populations in either trial.
Discussion
Bacterial population levels have not been established for cultivar responses with this hosUpathogen sys tem. High bacterial population levels can be found in resistant S. tuberosum cultivars; however, these are probably tolerant rather than resistant (Bonde et al., 1942; Manzer & McKenzie, 1988). Among the wild species, there seem to be four responses: immuni ty, high resistance, low resistance, and susceptibiIi-
473327.4 473327.5 473327.6 473327.8 473327.13 473327.15 473327.17
Age of mother clone 2
4 Months prr3 IFU/g:rX
4/5
X
0/4
0/5 0/11 0/10 0/10 0/5 4114
10' 10 1 0.2 X 10 1 0.2 x 10 1 0.1 x 10 1 0.0 0.0 0.0 0.0 0.0 0.3 X WI
7/10 2/8
0.8 X 10 1 OJ x 10 1
Oil 0
0.0 0.0 0.2
3/5 l/4 4/15 4110 2/10
0/6 115 0/5 4/10
0.6 1.0
x
0.0 0.8 x
101
101 1
8/13 10/11
0.8 x 10 0.0 0.6 x 10 1 1.0 x 10 1 1.0 x 10 1
8/13
1.1 x to'
7/10
Oil 2 6/10
6 Months
prr IFU/g
0114
0.6 0.0
x
10'
x x
10 1 10'
2/tO
0.0 0.1 0.2
0/5 0111 0/10
0.0 0.0 0.0
OliO
0.0 0.0 0.5 x 10 1 0.5 x 101
3110
0/5 8/14 5/10 2/10 4/10 2/6 2/5 015 0110
0.1 x 10' OJ x 10' 0.1 x 10 1 0.2 X 101 0.0 0.0· 0.2 X 10 1• 0.1 x 101
2/10 1/12 0/10
0.0·
7/13 3/ll 0113
1.2 X 10' 0.2 x to'· 0.0·
Plant introduction number.plant number.
Age of mother clone when cuttings were inoculated.
3 Plants with a positive IFAS result/total plants inoculated.
4 Mean immunofluorescing units per g tissue.
• Differs significantly for time effect (P =' 0.05), LS means. I
2
ty. Immune responses allow no bacterial reproduction or entry and, therefore, would give a negative IFAS reading. Nonimmune plants may give a resistant or susceptible response. Plants with high levels of resis tance would exhibit low bacterial populations, indicat ing a reduction in colonization of the vascular tissue. Susceptibility would occur when high bacterial popu lations are detected and plants exhibit typical symptom expression of ring rot. Low levels of resistance (high C. m. subsp. sepedonicus population levels with reduced symptom expression) also may occur. Environmental interactions may have resulted in the variation observed among the immune test clones evaluated at several different periods. Clones main
-131 tained in the greenhouse for extended lengths of time experience environmental changes of tempera ture, light, humidity, and moisture. The conditions under which the mother clones were maintained could have influenced the plant response during inoculation. A higher mean ring rot severity occurred when inocu lated 'Red Pontiac' plants were grown under low light intensity, and the total light energy under which the mother plants were grown may influence the response during inoculation (Nelson & Kozub, 1983). In host-virus pathogen systems, Matthews (1991) listed several environmental factors that may influence the response of the plant and may be applicable to host bacteria pathogen systems. Plants that are grown under high light intensity may exhibit reduced susceptibili ty, and plants that are grown under low light intensi ty may give an increased response. The time of the year at which plants are inoculated may also influence the plants' response. All plants were kept under low light intensity during the inoculation period. However, the high light intensity under which the mother clones were kept to inhibit tuberization may have influenced the response of the cuttings to inoculation (Nelson & Kozub, 1983). Apparent immunity in the five diploid species (S. infundibuliforme, S. lesteri, S. megistacrolobum, S. sanctae-rosae, and Group Stenotomum) may have resulted from failure of the root mass to be properly inoculated. Alternatively, they may have had a young seedling form of resistance. S. sanctae-rosae gave an immune response in the 19-month trial, so adult plant immunity may exist in this diploid species. During subsequent trials of the remaining diploid species, no immune response was found. However, the 19-month evaluation resembled the 15-month evaluation in the tetraploid S. acaule accessions, indicating that adult resistance may be present. The tetraploid genotype may have been more buffered against environmental fluctuations than the diploid genotype. Immunity is probably present for up to eight months in some S. acaule accessions (210029, 472643, 472651,472655, and 473327). Kurowski & Manzer (1992) also found S. acaule to be a good potential source of immunity. Greenhouse-maintained clones and clones grown from tubers performed well dur ing this period. This species should to be evaluated for host-pathogen interaction response during a nor mal annual growing season. Clones that contained low bacterial populations (less than 1.0 x 102 IFU/g) at the 15-month trial and an immune response in the 19-month trial may also be
immune plants. These clones, 472643.1, 472651.22, 472655.8, and 473327.7, may be a good source of immunity for introgression into the cultivated pota to. Several of these clones were also immune when regrown from tubers and tested at four and six months of age. Clones that gave a low bacterial population at both the 15- and 19-month evaluations, 472640.5, 472651.28, and 472655.4, may be highly resistant plants. Symptomless carriers are potentially hazardous to the potato industry (Bonde et al., 1942; Manz er & McKenzie, 1988). Thus, to avoid breeding of these carriers, ring rot screening should be done ear ly in the breeding program when introgression of this germplasm into domesticated S. tuberosum is being performed.
Acknowledgements
This research was supported in part by U.S. Department of Agriculture grant # 58-3K47-9-018. Germplasm was provided by the IR-l Potato Intro duction Station. Published with the approval of the director of the North Dakota Agricultural Experiment Station as journal article 12209.
References Baer, D. & N.C. Gudmestad, 1991. Serological detection of nonmu coid strains of Clavibacter michiganensis subsp. sepedonicus in potato. Phytopathology 82: 157-163. Bishop, A.L. & SA Slack, 1981. Population levels of Corynebac terium sepedonicum and symptom development of ring rot in potato plants. Phytopathology 71: 861. Bishop, A.L. & SA Slack, 1987. Effect of inoculum dose and preparation, strain variation, and plant growth conditions in the eggplant assay for bacterial ring rot. Am. Potato l. 64: 227-234. Bonde, R., F.l. Stevenson, c.F. Clark & R.V. Akeley, 1942. Resis tance of certain potato varieties and seedling progenies to ring rot. Phytopathology 32: 813-819. De Boer, S.H. & A. Wieczorek, 1984. Production of monoclonal antibodies to Corynebacteriumsepedonicum. Phytopathology 74: 1431-1434. De Boer, S.H. & M.E. McNaughton, 1986. Evaluation ofimmunoflu orescence with monoclonal antibodies for detecting latent bacte rial ring rot infections. Am. Potato l. 63: 533-543. De Boer, S.H. & M. McCann, 1990. Detection of Corynebacterium sepedunicum in potato cultivars with different propensities to express ring rot symptoms. Am. Potato l. 67: 685--694. Gudmestad, N.C., D. Baer & C,J. Kurowski, 1991. Validating immunoassay test performance in the detection of Corynebac terium sepedonicum during the growing season. Phytopathology 81: 475-480. Gutbrod, 0., 1987. Certification policies and practices in reference to bacterial ring rot. Am. Potato 1. 64: 677--681.
132 Hanneman, R.E. Jr. & J.B. Bamberg, 1986. Inventory of tuber bearing species. Wis. Agric. Exp. Sta. Bull. 533. Madison. 10hnston, S.A., T.P.M. denNijs, S.1. Peloquin & RE. Hanneman Jr., 1980. The significance of genic balance to endosperm develop ment in interspecific crosses. Theor. Appl. Genet. 57: 5-9. Kurowski, C.1. & F.E. Manzer, 1992. Reevaluation of Solanum species accessions showing resistance to bacterial ring rot. Am. Potato J. 69: 289-297. Logsdon, C.E., 1967. Effect of soil temperature on potato ring rot. Am. Potato J. 25: 281-286. Manzer, EE. & A.R McKenzie, 1988. Cultivar response to bacterial ring rot infection in Maine. Am. Potato J. 65: 333-339. Matthews, R.E.E, 1991. Plant Virology. Academic Press, Inc. New York. Miller, H.1., 1984. A method for the detection of latent ringrot in potatoes by immunofluorescence microscopy. Potato Res. 27: 33-42. Nelson, G.A., 1982. Corynebacteriumsepedonicumin potato: effect of inoculum concentration on ring rot symptoms and latent infec tion. Can. J. Plant Pathol. 4: 129-133. Nelson, G.A. & R.R. Harper, 1973. Factors affecting ring rot devel opment in root-inoculated potato plants originating from stem cuttings. Am. Potato J. 50: 365-370.
Nelson, G.A. & 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. Nelson, G.A. & B.c. Kozub, 1987. Effect of temperature and latent viruses on atypical ring rot symptoms of Russet Burbank Pota toes. Am. Potato J. 64: 589-597. Nelson, G.A., W.E. Torfason & F.R Harper, 1971. Comparison of inoculation methods in ring rot development in potato. Am. Potato 1. 48: 225-229. Racicot, H.N., D.B.G. Savile & I.L. Conners, 1938. Bacterial wilt and rot of potatoes-some suggestions for its detection, verifica tion, and control. Am. Potato 1. 15: 312-318. Schaad, MW. (ed.), 1980. Lab Guide for Identification of Plant Pathogenic Bacteria. Ann. Phytopathol. Soc., St. Paul, Minn. Sherf, A.E, 1949. Root inoculation, a method insuring uniform rapid symptom development of bacterial ring rot of potato. Phy topathology 39: 507-508. Spicer, P.B., 1961. Use of gibberellin to hasten germination of Solanum seed. Nature 189: 327-328. Sun, M.K.C. & G.D. Easton, 1983. Symptom expression of Corynebacterium sepedonicum-infected Russet Burbank potato plants in Montana. Am. Potato J. 60: 822.
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