Corynebacterium Sepedonicum Research Articles

Bacterial ring rot of potato caused by Clavibacter sepedonicus: Asuccessful example of defeating the enemy under international regulations.

BackgroundBacterial ring rot of potato (Solanum tuberosum) caused by the gram‐positive coryneform bacterium Clavibacter sepedonicus is an important quarantine disease threatening the potato industry around the globe. Since its original description in 1906 in Germany, management of ring rot has been a major problem due to the seedborne nature (via seed tubers not true seeds) of the pathogen allowing the bacterium to be transmitted long distances via infected tubers.Disease symptomsOn growing potato plants: interveinal chlorosis on leaflets leading to necrotic areas and systemic wilt. On infected tubers: vascular tissues become yellowish brown with a cheesy texture due to bacterial colonization and decay.Host rangePotato is the main host of the pathogen, but natural infection also occurs on eggplant, tomato, and sugar beet.Taxonomic status of the pathogenClass: Actinobacteria; Order: Actinomycetales; Family: Microbacteriaceae; Genus: Clavibacter; Species: Clavibacter sepedonicus (Spieckermann and Kotthoff 1914) Li et al. 2018.Synonyms (nonpreferred scientific names) Aplanobacter sepedonicus; Bacterium sepedonicum; Corynebacterium sepedonicum; Corynebacterium michiganense pv. sepedonicum; Clavibacter michiganensis subsp. sepedonicus.Microbiological propertiesGram‐positive, club‐shaped cells with creamy to yellowish‐cream colonies for which the optimal growth temperature is 20–23°C.DistributionAsia (China, Japan, Kazakhstan, Nepal, North Korea, Pakistan, South Korea, Uzbekistan, the Asian part of Russia), Europe (Belarus, Bulgaria, Czech Republic, Estonia, Finland, Georgia, Germany, Greece, Hungary, Latvia, Lithuania, Norway, Poland, Romania, European part of Russia, Slovakia, Spain, Sweden, Turkey, Ukraine), and North America (Canada, Mexico, USA).Phytosanitary categorizationCORBSE: EPPO A2 list no. 51. EU; Annex designation I/A2.

Upcoming Plant Pathological Techniques in the Disease Diagnosis

Serodiagnostics for the detection of plant pathogens particularly the plant viruses started in the fifties and had a quantum jump in application after the introduction of enzyme conjugated antibodies in enzyme linked immunosorbent assays EIA in the s Improvements and modifications in the original EIA made its application routine in the large scale indexing of vegetatively cultivated crops It is still the methods of choice for many purposes However its limited sensitivity as compared to nucleic acid based diagnostics particularly PCR restricts its use in critical decision making related to pathogens of quarantine significance like ring rot of potato caused by Corynebacterium sepedonicum tuber necrosis strains of PVY PVYNTN etc Serodiagnostics due to limited sensitivity and specificity in the detection of fungal and bacterial pathogens has not become popular for the detection of many of these pathogens Recent developments in the field of genomics of microorganisms and PCR based assays of microbes have revolutionized diagnostics A combination of immunocapture of pathogen and PCR has made the detection simpler and more sensitive than PCR alone Combination of immunocapture with multiplex PCR further reduces cost of detection Combination of nucleic acid probes and ELISA has the potential of its adoption for large scale indexing of propagating stocks WTO agreements related to trade in agricultural commodities need internationally acceptable diagnostic tools for the detection of pathogens of quarantine significance An internationally recognized monitoring organization is the urgent need The organization will be responsible for the evaluation of reagents and protocols for diagnostics and their regular update The issue was mooted in a recent symposium titled Moving Plant Disease Detection from the Ivory Towers to the Real World where a ring test process was described to validate and standardize new diagnostic procedures As more and more techniques are validated by the scientific community and their reliability and cost effectiveness demonstrated molecular based techniques will play a greater and more important role in the detection of pathogens

Clavibacter michiganensis subsp. sepedonicus

Clavibacter michiganensis<i> subsp. </i>sepedonicus

NADPH-dependent reductive ortho dehalogenation of 2,4-dichlorobenzoic acid in Corynebacterium sepedonicum KZ-4 and Coryneform bacterium strainNTB-1 via 2,4-dichlorobenzoyl coenzyme A

Corynebacterium sepedonicum KZ-4, described earlier as a strain capable of growth on 2,4-dichlorobenzoate (G.M. Zaitsev and Y.N. Karasevich, Mikrobiologiya 54:356-369, 1985), is known to metabolize this substrate via 4-hydroxybenzoate and protocatechuate, and evidence consistent with an initial reductive dechlorination step to form 4-chlorobenzoate was found in another coryneform bacterium, strain NTB-1 (W.J.J. van den Tweel, J.B. Kok, and J.A.M. de Bont, Appl. Environ. Microbiol. 53:810-815, 1987). 2-Chloro-4-fluorobenzoate was found to be converted stoichiometrically to 4-fluorobenzoate by resting cells of strain KZ-4, compatible with a reductive process. Experiments with cell extracts demonstrated that Mg - ATP and coenzyme A (CoA) were required to stimulate reductive dehalogenation, consistent with the intermediacy of 2-chloro-4-fluoro-benzoyl-CoA and 2,4-dichlorobenzoyl-CoA thioesters. 2,4-Dichlorobenzoyl-CoA was shown to be converted to 4-chlorobenzoyl-CoA in a novel NADPH-dependent reaction in extracts of both KZ-4 and NTB-1. In addition to the ligase and reductive dehalogenase activities, hydrolytic 4-chlorobenzoyl-CoA dehalogenase and thioesterase activities, 4-hydroxybenzoate 3-monooxygenase, and protocatechuate 3,4-dioxygenase activities were demonstrated to be present in the soluble fraction of KZ-4 extracts following ultracentrifugation. We propose that the pathway for 2,4-dichlorobenzoate catabolism in strains KZ-4 and NTB-1 involves formation of 2,4-dichlorobenzoyl-CoA, NADPH-dependent ortho dehalogenation yielding 4-chlorobenzoyl-CoA, hydrolytic removal of chlorine from the para position to generate 4-hydroxybenzoyl-CoA, hydrolysis to form 4-hydroxybenzoate, oxidation to yield protocatechuate, and oxidative ring cleavage.

Characterization of a pathogen-induced potato catalase and its systemic expression upon nematode and bacterial infection.

We have isolated a cDNA encoding a catalase (Cat2St) by differential screening of a cDNA library constructed from potato roots infected with the cyst nematode Globodera pallida. Expression analysis confirmed the local induction of Cat2St and showed that it was highest at the adult stage of the parasite. It also revealed that Cat2St was induced in uninfected roots, stems, and leaves of infected plants. Localized and systemic induction of Cat2St was also observed upon root-knot nematode (Meloidogyne incognita) and root bacteria (Erwinia carotovora, Corynebacterium sepedonicum) infections. Based on sequence and expression analysis, Cat2St was found to belong to the recently described class II of dicotyledonous catalases, suggesting that these catalase isoforms could also be pathogen induced. Plant-parasitic nematodes are known to induce, in the roots of their hosts, highly metabolic feeding cells that function as nutritional sinks. Whereas the local induction of Cat2St is probably a consequence of an oxidative stress of metabolic nature, the systemic induction of Cat2St shows striking similarities with the induction of systemic acquired resistance (SAR) genes. The possible role of catalase in compatible plant-pathogen interactions is discussed.

Identification of Clavibacter michiganensis subsp. sepedonicus using the polymerase chain reaction.

From the sequence of a subcloned DNA fragment of a highly conserved plasmid of Clavibacter michiganensis subsp. sepedonicus a pair of oligonucleotides were devised for use as polymerase chain reaction primers. The primer sequences do not show significant homology with any other sequence deposited in public databases. Polymerase chain reactions carried out using this primer pair and untreated cells of all strains of C. michiganesis sepedonicus tested resulted in the amplification of a DNA fragment of about 670 base pairs. No amplification was observed when bacteria belonging to other species were submitted to polymerase chain reaction under the same conditions. The detection limit of the assay was 4 x 10(3) bacteria.

Ring rot symptom development on potato cultivars and lines in southern Alberta

Plants were grown in the field from seed pieces of potato cultivars injected with ring rot bacteria (Corynebacterium sepedonicum). Symptoms were produced with both 18 and 180 colony forming units (cfu) on cvs Norchip and Red Pontiac and with 180 cfu on cv. Belrus plants. Tuber symptoms were detected in all cultivars except Belrus and Teton. Tuber progeny produced plants that developed top symptoms on all cultivars except those of cv. Nooksack, Russet Burbank, and Teton. Tuber progeny of the second crop produced both plants and tubers with symptoms developing only in the cv. Nooksack. In another 3-year experiment, variability in the disease response of selected cultivars and lines was examined following knife-inoculation of tuber seed with a high level of ring rot bacteria. Significant correlations between top and tuber symptoms were detected, but they were not high enough to make unnecessary the examination of both top and tuber symptoms in ring rot disease selection studies.

Genetic control of degradation of chlorinated benzoic acids in Arthrobacter globiformis, Corynebacterium sepedonicum and Pseudomonas cepacia strains

The strains of Arthrobacter globiformis KZT1, Corynebacterium sepedonicum KZ4 and Pseudomonas cepacia KZ2 capable of early dehalogenation and complete oxidation of 4-chloro-, 2,4-dichloro-and 2-chlorobenzoic acids, respectively, have been analyzed for the origin of the genetic control of degradation. The occurrence and molecular sizes of plasmids in all the strains have been established. Plasmid pBS1501 was shown to control 4-chlorobenzoate dehalogenation in the case of KZT1 strain. The same possibility is proposed for plasmid pBS1502 for dehalogenation of 2,4DCBA by KZ4 strain. The chromosome localization of the genes controlling oxidation of 4-hydroxybenzoate in strain KZT1 is shown. Localization of the whole set of genes responsible for 2CBA degradation in the strain KZ2 chromosome is suggested.

Genetic control of degradation of chlorinated benzoic acids inArthrobacter globiformis, Corynebacterium sepedonicumandPseudomonas cepaciastrains

The strains of Arthrobacter globiformis KZT1, Corynebacterium sepedonicum KZ4 and Pseudomonas cepacia KZ2 capable of early dehalogenation and complete oxidation of 4-chloro-, 2,4-dichloro-and 2-chlorobenzoic acids, respectively, have been analyzed for the origin of the genetic control of degradation. The occurrence and molecular sizes of plasmids in all the strains have been established. Plasmid pBS1501 was shown to control 4-chlorobenzoate dehalogenation in the case of KZT1 strain. The same possibility is proposed for plasmid pBS1502 for dehalogenation of 2,4DCBA by KZ4 strain. The chromosome localization of the genes controlling oxidation of 4-hydroxybenzoate in strain KZT1 is shown. Localization of the whole set of genes responsible for 2CBA degradation in the strain KZ2 chromosome is suggested.

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.

Validating Immunoassay Test Performance in the Detection ofCorynebacterium sepedonicumDuring the Growing Season

The growth and serological detection of Corynebacterium sepedonicum in potato stems was studied in the growth chamber and in the field. Populations of C. sepedonicum were similar in two susceptible cultivars, Norchip and Russet Burbank, which differ in seasonal maturity. The maximum population of the ring rot bacterium in these cultivars was between 10 8 and 10 9 cfu/g fresh weight, depending on the growing season. Populations of C. sepedonicum in stems of field-grown plants of Belrus developed more slowly and were generally lower when compared to the two susceptible cultivars. Development of bacterial ring rot symptoms in Norchip and Russet Burbank was associated with bacterial populations ≥10 7 cfu/g fresh weight (...)

Elimination of bacterial ring rot (Corynebacterium sepedonicum [Spieck. and Kotth.] Skapt. and Burkh.) by in vitro culture of sprout tissue

Apical explants from sprout tissue, cultured in vitro, of potato tubers (cv. Russet Burbank) infected withCorynebacterium sepedonicum were demonstrated to be free of the pathogen. The material remained free of the pathogen through three in vitro generations and through two tuber generations produced from the in vitro plantlets.

Detection of bacterial cell wall hydrolases after denaturing polyacrylamide gel electrophoresis.

Cell walls from various Gram-positive bacteria were incorporated at a concentration of 0.2% (w/v) into polyacrylamide gels as a substrate for detection of cell wall hydrolases. Bacterial extracts from crude cell wall preparations were denatured with sodium dodecyl sulfate and 2-mercaptoethanol and subjected to denaturing polyacrylamide gel electrophoresis in gels containing bacterial cell walls. After renaturation in the presence of purified and buffered 1% (v/v) Triton X-100, cell wall hydrolases were visualized as clear lytic zones against the opaque cell wall background. One to fifteen bands with lytic activity could be detected, depending on bacterial extracts and on the nature of the cell walls incorporated into gels. Crude cell wall extracts were the best source of cell wall hydrolases from various Gram-positive bacteria such as Clostridium perfringens (15 bands), Micrococcus luteus (1 band), Bacillus megaterium (4 bands), Bacillus sp. (6 bands), B. cereus (3 bands), B. subtilis (7 bands), Staphylococcus aureus (13 bands), Streptococcus faecalis (3 bands), and Strep. pyogenes (5 bands). Molecular masses of cell wall hydrolases ranged from 17 to 114.6 kDa. Lytic activities against cell walls of Corynebacterium sepedonicum (Clavibacter michiganense pv. sepedonicum) could be shown with the cell wall extracts of Strep. pyogenes (45.7 kDa), Strep. faecalis (67 kDa), B. megaterium (67 kDa), and Staph. aureus (67 kDa).

Determination of Population Densities ofCorynebacterium sepedonicumin Potato Stems During the Growing Season

Within 80 days after planting, C. sepedonicum could be detected in symptomless stems of all three cultivars by immunofluorescence with a cell wall-specific monoclonal antibody and by enzyme-linked immunosorbent assay with an extracellular polysaccharide-specific monoclonal antibody. The ELISA values were positively correlated (correlation coefficient=0.85) with estimates of population densities

An automated microscope system for estimating the population of Corynebacterium sepedonicum cells labelled with monoclonal antibodies in immunofluorescence

An automated microscope system for estimating the population of Corynebacterium sepedonicum cells labelled with monoclonal antibodies in immunofluorescence

Characterization of two DNA clones specific for identification of Corynebacterium sepedonicum

Escherichia coli TB1 was transformed with pUC9 containing fragmented DNA (4–10 kilobases (kb)) from Corynebacterium sepedonicum. The resulting genomic bank was screened by a dot blot assay to identify clones specifically hydridizing to C. sepedonicum DNA and not to the DNA of several other Gram-positive and Gram-negative bacteria. Two clones (III24 and III31) were selected because of their ability to strongly hybridize to C. sepedonicum DNA and weakly hybridize to the DNA of C. michiganense, Erwinia carotovora, Agrobacterium tumefaciens, Bacillus subtilis, Pseudomonas solanacearum, Micrococcus luteus, and Arthrobacter globiformis. These two clones were also specific for C. sepedonicum DNA when tested against the DNA from 30 isolates of soil bacteria. Restriction enzyme analysis has shown that the two clones have an insert of 8 kb (III24) and 4 kb (III31). On the basis of restriction enzyme patterns, one clone (III24) does not correspond to plasmid pCL 50, a cryptic plasmid found in several C. sepedonicum isolates. Because purified III24 and III31 DNA can be used to detect approximately 1 ng of C. sepedonicum genomic DNA, the two clones can complement serological or biological detection methods. This could be useful, especially when a high degree of specificity is required for detection or identification of this plant pathogen.

Effect of temperature and latent viruses on atypical ring rot symptoms of Russet Burbank potatoes

The ring rot bacterium,Corynebacterium sepedonicum (Spieck. and Kotth.) Skapt. and Burkh. [Clavibacter michiganense subsp.sepedonicum (Spieck et Kotth.) Davis et al.], and latent potato viruses (potato virus S and potato virus X) were investigated for their effect on atypical (ATYP) ring rot symptom development on Russet Burbank potato plants at different temperatures. Plants grown at 21 C from stem cuttings root-inoculated withC. sepedonicum developed typical wilting and chlorotic symptoms of ring rot that were equally severe on virus-free (VF) and virus-infected (VI) plants. All VF and VI plants grown at 15 C from inoculated stem cuttings exhibited ATYP symptoms that included extreme stunting, resetting, and chlorotic symptoms of ring rot. More severe ATYP symptoms developed on VI than on VF plants. Up to 5 wk after inoculation withC. sepedonicum, ATYP symptom severity ratings of both VF and VI plants increased and declined thereafter. The ATYP severity ratings were highly correlated with fresh weight of plants with high severity ratings being associated with low fresh weights. These results emphasize the need to determine the role of temperature and viral pathogens on ring rot symptomology in existing and newly developed potato cultivars and thereby enable better field detection of bacterial ring rot.

Homology of pCS1 Plasmid Sequences with Chromosomal DNA in Clavibacter michiganense subsp. sepedonicum : Evidence for the Presence of a Repeated Sequence and Plasmid Integration

Restriction fragments of pCS1, a 50.6-kilobase (kb) plasmid present in many strains of Clavibacter michiganense subsp. sepedonicum ("Corynebacterium sepedonicum"), have been cloned in an M13mp11 phage vector. Radiolabeled forms of these cloned fragments have been used as Southern hybridization probes for the presence of plasmid sequences in chromosomal DNA of this organism. These studies have shown that all tested strains lacking the covalently closed circular form of pCS1 contain the plasmid in integrated form. In each case the site of integration exists on a single plasmid restriction fragment with a size of 5.1 kb. Southern hybridizations with these probes have also revealed the existence of a major repeated sequence in C. michiganense subsp. sepedonicum. Hybridizations of chromosomal DNA with deletion subclones of a 2.9-kb plasmid fragment containing the repeated sequence indicate that the size of the repeated sequence is approximately 1.3 kb. One of the copies of the repeated sequence is on the plasmid fragment containing the site of integration.

Interpretation of the EC method for the detection of latent Corynebacterium sepedonicum infections in potato1

The EC method for the detection of latent ring‐rot infections (Corynebacterium sepedonicum) consists essentially of an indirect immunofluorescence test and a pathogenicity test on eggplant (EP). When interpreting the results of this method, care should be taken that: (a) eggplants are grown at 21°C. At 28–29°C the detection level of the EP was increased 102 to 103‐fold to 105 to 106 cells ml‐1 and latent infections (which may go unnoticed) occurred with 104 to 105 cells ml‐1; (b) reisolations are made from symptomless eggplants grown at the optimum temperature (21°C) as latent infections can also occur in these plants at concentrations of 102 to 103 cells ml‐1; (c) potato tuber extracts are tested without freezing or stored freeze‐dried or in a proper protective agent. Freezing at ‐20°C in maceration buffer of cells of C. sepedonicum and Pseudomonas solanacearum (the latter was included for comparison) reduced their numbers by 90–98% after one day. This could easily cause viable cell numbers to drop below the detection level of the pathogenicity test.

Methods for detection of the blackleg, ring rot and gangrene pathogens in potato nuclear stock mother tubers and plantlets1

Methods are described for the detection of latent infection by Erwinia carotovora ssp. atroseptica (blackleg and soft rot), Corynebacterium sepedonicum (ring rot) and Phoma exigua vat. foveata (gangrene) which are regarded as the most important tuber‐borne pathogens of potato. The methods comprise selective media for E. carotovora ssp. atroseptica and P. exigua var. foveata, indirect fluorescence antibody staining for E. carotovora ssp. atroseptica and C. sepedonicum, and the eggplant test for C. sepedonicum and P. exigua var. foveata. These are used in Northern Ireland to detect pathogen contamination in nuclear stock mother tubers prior to micropropagation. Marked reductions in disease levels of blackleg have been observed in present pre‐basic stocks raised by micropropagation compared with those previously propagated from stem cuttings. It was found that Pseudomonas stutzeri, a saprophytic contaminant bacterium, can pass through the micropropagation process in a latent form.