Bacterial Ring Rot Research Articles

Immunity to Clavibacter michiganensis subsp. sepedonicus: Inheritance of immunity in Solanum acaule

Bacterial ring rot of potato (Solanum tuberosum) has not been successfully controlled through management of certified seed. Therefore, the identification of immunity to this pathogen and its introgression into the cultivated potato is vitally important to the potato industry. Immunity was detected in the disomic tetraploid 2EBN (Endosperm Balance Number) species S. acaule. Immune and nonimmune parents were crossed in a 4 × 4 mating scheme that consisted of four immune × immune crosses, four immune × nonimmune, four nonimmune × immune, and four nonimmune × nonimmune crosses. Analysis was performed on the 16 F1 populations and 54% of the progeny was found to exhibit an immune response to inoculation with Clavibacter michiganensis subsp. sepedonicus (Spieck & Kotth.) Davis et al. Immunity appears to be conferred by two dominant alleles, one at each locus, and may be associated with minor or modifying genes. Phenotypic expression of nonimmune progeny ranged from susceptible to resistant, probably due to minor or modifying genes. These results indicate that it may not be difficult to transfer immunity into the cultivated potato.

Detection and identification of Clavibacter michiganensis subsp. sepedonicus and Clavibacter michiganensis subsp. michiganensis by nonradioactive hybridization, polymerase chain reaction, and restriction enzyme analysis

To develop a rapid and reliable detection and identification method for Clavibacter michiganensis subsp. sepedonicus and C. michiganensis subsp. michiganensis, two biotinylated probes and derived primer sets were evaluated for specificity using a large number of bacterial strains. Detection in dot blot analysis using the Diagen probe against C. michiganensis subsp. sepedonicus was possible with all 32 C. michiganensis subsp. sepedonicus strains tested. Cross-hybridization occurred with all nine C. michiganensis subsp. insidiosus strains tested. No hybridization occurred with any of 54 other related and unrelated bacterial strains including C. michiganensis subsp. michiganensis, C. michiganensis subsp. nebraskensis, C. michiganensis subsp. tessellarius, C. iranicus, C. rathayi, and C. tritici and potato saprophytes. Hybridization of the MIC 1 probe against C. michiganensis subsp. michiganensis was obtained with 22 out of 24 C. michiganensis subsp. michiganensis strains. A weak hybridization signal occurred only with two strains of C. michiganensis subsp. insidiosns. No hybridization occurred with any of the 71 other related and unrelated bacterial strains tested including tomato saprophytes. Restriction fragment length polymorphisms detected with the Diagen probe allowed differentiation between C. michiganensis subsp. sepedonicus and the related C. michiganensis subsp. insidiosus. Restriction fragment length polymorphism analysis using the MIC 1 probe and BamH1 showed at least two groups of patterns within C. michiganensis subsp. michiganensis. By using a primer set derived from the Diagen probe, a DNA sequence could be amplified with all C. michiganensis subsp. sepedonicus strains tested. Only the nontarget organism C. michiganensis subsp. insidiosus yielded a similar polymerase chain reaction product. Restriction enzyme analysis of the polymerase chain reaction product enabled rapid distinction between the subspecies. With a CMM primer set derived from the MIC 1 probe a DNA sequence was amplified from the same 22 out of 24 C. michiganensis subsp. michiganensis strains that showed hybridization with the MIC 1 probe. The polymerase chain reaction product could be verified by restriction enzyme analysis. The Diagen and MIC 1 probes and derived primer sets were shown to be useful for the detection and identification of C. michiganensis subsp. sepedonicus and C. michiganensis subsp. michiganensis. The MIC 1 probe, however, failed to detect two strains of the latter subspecies.Key words: biotin, PCR, REA, potato bacterial ring rot, bacterial canker of tomato, RFLP, Clavibacter michiganensis subsp. insidiosus.

Comparison of some diagnostic assays for bacterial ring rot of potato: A case study

The results of testing 48 stems and tuber samples, representing 15 seed lots from several different growers, for the presence of the causal organism of bacterial ring rot,Clavibacter michiganensis subsp.sepedonicus with the gram stain, latex agglutination (LAT), enzyme-linked immunosorbent assay (ELISA), and a DNA hybridization assay (DHA) were compared. The frequency with which positive tests were obtained varied significantly among assays and was associated with symptom expression in samples. Detection was highest with the serological assays and lowest for DHA. Use of the gram stain, in conjunction with either LAT, ELISA, or DHA reduced the number of positive diagnoses, but increased the agreement between results for different assay combinations. Based on a comparison of results for both individual samples and seed lots, the performance of LAT and ELISA for diagnostic purposes were equivalent. DHA was found to be less useful due to limitations in test sensitivity forC. m. sepedonicus, but it does give unequivocal diagnoses of samples where bacterial populations are sufficient to allow detection.

Identification of parents suitable for molecular mapping of immunity and resistance genes in solanum species

A survey of DNA polymorphisms and bacterial ring rot reactions inSolanum species was conducted to identify parents that when crossed would produce an F1 mapping population useful for identifying DNA markers tightly linked to genes for immunity or resistance to bacterial ring rot.Solanum tuberosum, S. acaule, diploidSolanum species, doubled monoploid lines ofS. phureja, and somatic fusion hybrids betweenS. tuberosum andS. brevidens were established in tissue culture from single true potato seeds or tubers and evaluated for bacterial ring rot reactions in greenhouse studies with root-inoculated tissue cultured plantlets. Establishment of tissue culture stocks insured the availability of homogeneous genetic material for use in bacterial ring rot assays and genetic analyses. Presence ofClavibacter michiganense subsp.sepedonicum was assessed in stems by an immunofluorescence antibody assay (IFAS). Two accessions were found to be immune to bacterial ring rot. DNA polymorphisms were detected within and among 24 accessions evaluated with 24 RFLP markers and 10 arbitrary oligonucleotide primers (RAPDs). These results identify parents suitable for future mapping of gene (s) for immunity or resistance to bacterial ring rot.

AC Ptarmigan: An early maturing potato cultivar with good chipping and fresh market quality

AC Ptarmigan is an early maturing, buff skin, cream flesh potato cultivar which has good, stable chip quality and can be processed directly from the field. The cultivar has uniform attractive tubers with shallow eyes. It is moderately resistant to common scab, early blight, potato virus Y and potato leafroll virus. It is susceptible to late blight and shows clear field symptoms when infected with bacterial ring rot.

DETECTION OF BACTERIAL RING ROT OF POTATO USING DNA PROBE AND PCR

One hundred twenty-eight field samples of 25 potato stem sections were analyzed for detecting bacterial ring rot (BRR). Samples containing more than 105, between 105 and 104, and <104 immunofluorescing BRR cells per milliliter of sample, detected using immunofluorescence-antibody staining with MAb9A1, were used to compare the efficiency of two other detection methods. Samples were screened with a digoxigenin-labeled DNA probe (Bh1 2, 0.6 kbp) detected by chemioluminescence on nylon membrane. Samples also were screened with a PCR test using primers derived from the sequence of the Bh12 probe. DNA probe tests on these three bacterial concentrations showed a detection efficiency of 100%, 76.8%, and 8.0%, respectively, whereas detection efficiencies of 100%, 100%, and 84.5% were obtained with PCR tests. Almost all positive samples gave the expected 403 bases ethidium-bromide-stained band when the amplified products were analyzed on 1.4% agarose gel. Thus, the PCR test was a sensitive detection method for screening bacterial ring rot of potato.

Effect of Interaction of Inoculum Dose, Cultivar, and Geographic Location on the Magnitude of Bacterial Ring Rot Symptom Expression in Potato

The effect of the interaction of inoculum dose of Clavibacter michiganensis subsp. sepedonicus, potato cultivar, and geographic location on the magnitude of bacterial ring rot symptom expression was investigated by planting an inoculum dose X cultivar factorial experiment in several potato-growing regions of the United States. Inoculum dose was positively correlated with the expression of both foliar and external tuber symptoms of bacterial ring rot and negatively correlated with total yield and vine length. The magnitude of the response to inoculum dose was modulated primarily by its interaction with cultivar and location. Three-way interaction between inoculum dose, cultivar, and location was generally not significant [...]

Evaluation of Serological Tests for Detection ofClavibacter michiganensissubsp.sepedonicusin Composite Potato Stem and Tuber Samples

A procedure to index potato seed lots for bacterial ring rot was evaluated by testing composite samples of tissue from 200 potato stems or tubers, in blind experiments, for the presence of Clavibacter michiganensis subsp. sepedonicus by immunofluorescence and enzyme-linked immunosorbent assay (ELISA) in six laboratories. Different antibody preparations were used for the two serological tests. Tissue samples were from ring rot-free potato plants, but ring rot-infected tissue was added to 88 of the 385 composite samples to create authentic positive composites. One laboratory achieved 100% efficiency for detection of ring rot infections in stem samples with ELISA, but the overall sensitivity and specificity of ELISA were 97.7 and 90.1%, respectively, for stems and 93.3 and 86.7%, respectively, for tubers [...]

Effect of Interaction of Inoculum Dose, Cultivar, and Geographic Location on the Development of Foliar Symptoms of Bacterial Ring Rot of Potato

Proportional hazards models were used to estimate hazard and survival functions for both the onset and maximum incidence of foliar symptoms of bacterial ring rot in three potato cultivars (Norchip, Norland, and Russet Burbank) grown at seven locations across the United States (Colorado, Maine, New York, North Dakota, Oregon, Washington, and Wisconsin) over the period 1988-1990. The models predicted a minimum of 50 and 82 days after planting for survival probabilities of P<0.95 and P<0.05 for symptom onset and maximum disease incidence, respectively. The time frame predicted for these events was affected by cultivar, location, and cultivar by location interaction. There was no proportional increase in the relative hazards for symptom onset and maximum disease incidence due to increased inoculum dose [...]

Potato Flea Beetle (Coleoptera: Chrysomelidae) Evaluated as a Possible Vector of Ring Rot Bacterium in Potatoes

Commercial tolerances for the incidence of bacterial ring rot caused by Clavibacter michiganense subsp. sepedonicum (Spieck. & Kotth.), Davis, Gillaspie, Vidaver &Harris in potatoes, Solanum tuberosum L., are zero, requiring strict regulations to reduce field spread of the disease. Few insects have been shown to be directly associated with the dissemination of ring rot bacteria. Concentrations of bacteria within potato leaves as potential inoculum and the ability of the potato flea beetle, Epitrix cucumeris (Harris), to acquire and disseminate the ring rot bacterium were investigated. There was considerable variation in the concentrations of ring rot bacteria within potato leaves. The highest numbers of bacteria were found in the midveins. Fewest bacteria were present in the mesophyll of the laminae, but the basal sections of laminae contained more bacteria than did the midrib sections from leaflet tips. Potato flea beetles acquired the bacteria in 24 h or less. Beetles exposed to infected plant tissue were confirmed positive for the pathogen using indirect immunofluorescence antibody staining (IFAS). In addition, scanning electron photomicrographs of infected flea beetles showed their mouthparts to be contaminated with bacteria that were the same size and morphology as the ring rot pathogen. Positive IFAS reactions of potato test plants that had been fed upon by potato flea beetles previously exposed to ring rot-infected plants represented support for transmission of the bacteria by the beetles. Mode of transmission appears to be through feeding activity because IFAS analyses of regurgitants and fecal material from contaminated beetles were negative.

Detection of Clavibacter michiganensis subsp. sepedonicus by DNA amplification

Control of bacterial ring rot ultimately depends on the accurate and sensitive detection of C. michiganensis subsp. sepedonicus in infected potato tissues and tubers. Polymerase chain reaction (PCR) based detection of C. michiganensis subsp. sepedonicus appears to have the potential to circumvent many of the problems currently associated with detection of this phytopathogen. PCR reactions using primers specific to C. michiganensis subsp. sepedonicus and genomic DNAs from related strains, including phytopathogens, did not produce any amplification products. C. michiganensis subsp. sepedonicus was detectable from a mixture of potato and bacterial DNA by amplification of a DNA sequence specific to C. michiganensis subsp. sepedonicus. Detection by DNA amplification allowed direct processing of plant tissue samples, and circumvented the need for prior isolation of the suspected phytopathogen.

Comparison of a DNA Hybridization Probe and ELISA for the Detection ofClavibacter michiganensissubsp.sepedonicusin Field-Grown Potatoes

Clavibacter michiganensis subsp. sepedonicus, the causal agent of bacterial ring rot, was detected in field-grown potatoes using a 1.078-kb repeated C. m. sepedonicus sequence as a probe in DNA hybridizations. Stem and petiole samples from susceptible and tolerant cultivars (Russet Burbank and Belrus, respectively), inoculated with 10 mM phosphate buffer (pH 7.2) or 10 2 or 10 9 cfu of either an aggressive or a less aggressive C. m. sepedonicus strain, were processed by directly blotting cut tissue sections on nylon membranes, macerating frozen tissues, and applying xylem fluid collected by centrifugation to nylon membranes (stems only)

Symptomless infection with Clavibacter michiganensis subspecies sepedonicus during tissue culture propagation of potato

Nodal cuttings of tissue cultured potato (Solarium tuberosum L. ’Sangre’) were inoculated with the bacterial ring rot (BRR) pathogen, Clavibacter michiganensis subsp. sepedonicus (Cms). Under commercial tissue culturing conditions, the pathogen persisted through as many as three micropropagation cycles, each consisting of production of a plantlet from an apical cutting. Infected plantlets did not develop BRR symptoms of leaf wilt or chlorosis. Signs of bacterial presence, cloudiness of tissue culturing medium or foliar lesions, were not present when the inoculum level was low, 5.0 × 102 cfu mL−1. A higher inoculum concentration (5.0 × 103 cfu mL−1) reduced plantlet height, but the lower concentration had no effect on height. Growth and viability of tissue from Cms-inoculated plantlets grown on Murashige and Skoog’s medium without sucrose, a source of carbon, were reduced compared to those grown with sucrose, but BRR symptoms were not induced at these inoculum levels. When transplanted to soilless potting medium, a basal portion of cycle 1 plantlets either died or produced a symptomatic miniplant, indicating that Cms survived during the growth of a plantlet into a miniplant. Nonsymptomatic miniplants were produced by portions of some cycle 2 and 3 Cms-infected plantlets, indicating that successive nodal propagation decreased the number of symptomatic miniplants. Bioassays of these nonsymptomatic miniplants were Cms-negative. Incubating plantlet tissue in Richardson broth for 14 d and visually observing for turbidity was not effective in detecting Cms in cultured potato tissue. Because phytopathogenic bacteria may persist in tissue cultures without causing symptoms, pathogen-specific testing of source plants and plant cultures is necessary to prevent transmission of bacteria to cultured stock and field-grown progeny.Key words: Potato shoot cultures, Solanum tuberosum L., Clavibacter michiganensis subsp. sepedonicus, medium with no carbon, bacterial persistence and detection, symptomless infection, Solanum melongena L.

A comparative study in five laboratories on detection ofClavibacter michiganensis subsp.sepedonicus in potato stems and tubers

Potato stems and tubers grown in the field from seed tubers inoculated withClavibacter michiganensis subsp.sepedonicus, which causes bacterial ring rot, were tested by indirect, sandwich enzyme-linked immunosorbent assay (ELISA) in five laboratories. Correlation between values for each experimental treatment from the five laboratories was greater (r=0.86) than correlation between values for individual samples (r=0.71). When three or more laboratories obtained ELISA values of ≥0.200 for a sample, that sample was presumed to be positive. Conversely, when three or more laboratories obtained ELISA values <0.200, the consensus determination was regarded as negative. The percentage of stem and tuber samples that were in agreement with the consensus ELISA determination varied from 65.5 to 96.7%. Indirect immunofluorescence tests, conducted on the same samples in two laboratories, were consistent with 83.4–91.9% of the consensus ELISA determinations. Presence or absence ofC.m. sepedonicus was confirmed in some samples by an eggplant bioassay and direct isolatiion of the bacterium. The ELISA procedure was well suited for screening large numbers of samples and this study confirms it to be a promising procedure in routine indexing of seed potatoes forC.m. sepedonicus.

Bacterial ring rot disease development in resistant and susceptible cultivars

In two greenhouse tests, we compared cell populations ofCorynebacterium sepedonicum in inoculated cultivars which had failed to show symptoms in field inoculation tests against cultivars that produce well-defined symptoms. Both population studies showed that cell numbers as high as 1 × 109 colony-forming units per gm of fresh weight (cfu gm-1) were reached in some cultivars prior to symptom development. In one of these tests, conducted when day length averaged 14 1/2 hrs, some cultivars failed to show symptoms 11 wks after inoculation but were found to have cell populations as high as 3.2 × 108 cfu gm-1 7 wks after inoculation. In another study, root and seed piece inoculations were compared in susceptible and resistant cultivars and only minor differences in symptom expression due to mode of entry of the pathogen were found.

Reevaluation ofSolanum species accessions showing resistance to bacterial ring rot

One hundred and fifty-six plant accessions, including susceptible checks, from the IR-1 collection were evaluated for resistance/immunity to bacterial ring rot (BRR). Each accession selected had been previously rated as highly resistant to BRR based on symptom expression but, in our tests, 57 of them yielded no plants which failed to produce symptoms. However, the remaining 99, represented by a total of 2589 inoculated plants, yielded 1000 plants which failed to produce BRR symptoms after eight weeks. Immunofluorescent antibody stain testing showed that 679 of the symptomless plants supported detectable numbers ofCorynebacterium sepedonicum cells. Comparison of root-inoculated and tuber-inoculated accessions showed that root-inoculations yielded significantly fewer symptomless and bacterial cell-free plants. No significant differences were found in BRR reactions of fiveSolanum spp. when inoculated with two different strains ofC. sepedonicum. Appreciable numbers of symptomless plants were obtained from inoculated progeny of crosses with resistant cultivars but none of those retested proved to be immune.

Isolation and Characterization of Extracellular Polysaccharide ofClavibacter michiganensissubsp.sepedonicus

The extracellular polysaccharide (EPS) of Clavibacter michiganensis subsp. sepedonicus, the causal organism of bacterial ring rot of potato, consists of four components (I-IV) that were separated on the basis of size and charge. These components were homogenous according to ion-exchange and/or permeation chromatography, to analysis of neutral sugar composition, and by reaction with polyclonal antisera (...)

A Semiselective Agar Medium for Isolation ofClavibacter michiganensissubsp.sepedonicusfrom Potato Tissues

A new semiselective agar medium, NCP-88, was developed for isolating Clavibacter michiganensis subsp. sepedonicus, the cause of bacterial ring rot, from infected potato plant parts. Important selective components of this nutrient agar, yeast extract, and salts medium are mannitol, polymyxin B-sulfate, nalidixic acid, and cycloheximide. NCP-88 permitted a better balance between plating efficiency and selectivity than did other reported semiselective media (.)

Insect transmission of the bacterial ring rot pathogen

Three species of potato-infesting insects were evaluated under laboratory and field conditions for their capability to transmitCorynebacterium sepedonicum (Spieck. & Kotth.) Skapt. & Burkh., the bacterial ring rot (BRR) pathogen of potatoes. Laboratory and field studies confirmed the vector capability of the Colorado potato beetle (CPB),Leptinotarsa decemlineata (Say) (Coleoptera: Chrysomelidae) and the green peach aphid (GPA),Myzus persicae (Sulzer) (Homoptera: Aphididae). Both adults and larvae of the CPB proved to be vectors of the pathogen. Only adult GPA were evaluated. Bacterial transmission was most likely mechanical: analyses of the CPB mouthparts stained by the indirect immunofluorescence antibody staining (IFAS) procedure confirmed the presence of the bacterium after the beetles were exposed to infected plant tissue. Eggs, haemolymph, feces, regurgitations, and macerates of the digestive tract failed to show the presence of the bacterium. The aster leafhopper,Macrosteles fascifrons (St∮al) (Homoptera: Cicadellidae) failed to acquire and transmitCorynebacterium sepedonicum.

Current status and future prospects of bacterial ring rot testing

The bacterial ring rot disease of potato (Corynebacterium sepedonicum) can be detected in seed potato lots by serological tests with greater accuracy than by visual field inspections. Indexing tests can make use of highly specific immunofiuorescence probes, enzyme-linked immunosorbent assays, and perhaps other procedures. Sensitivity and specificity of these procedures need to be determined in order to estimate the confidence with which ring rot can be detected in a seed lot.