scholarly journals Sensitivity and specificity of monoclonal antibody Mn-Cs1 for detection and determination of Clavibacter michiganensis subsp. sepedonicus, the causal agent of bacterial ring rot of potato

2012 ◽  
Vol 38 (No. 4) ◽  
pp. 117-124 ◽  
Author(s):  
I. Pánková ◽  
B. Kokošková
Keyword(s):  
Monoclonal Antibody ◽  
Sensitivity And Specificity ◽  
Extracellular Polysaccharides ◽  
Bacterial Strains ◽  
Clavibacter Michiganensis ◽  
Bacterial Ring Rot ◽  
Clavibacter Michiganensis Subsp ◽  
Homologous Antigen ◽  
High Level

Monoclonal antibody Mn-Cs1 with a high level of sensitivity and specificity for detection and determination of Clavibacter michiganensis subsp. sepedonicus was prepared. Strain C. m. subsp. sepedonicus NCPPB 3467 (as whole cell antigen and extracellular polysaccharides) was used for immunisation of four mice Balb/c. After cloning and verifying, two stable hybridoma clones were gained. One monoclonal antibody, designated Mn-Cs1, was used in all tests. It reacted intensely with extracellular polysaccharides from homologous antigen (> 0.5 mg/ml), weakly with proteins from cell walls (> 200 μg/ml) and with whole homologous antigen (concentration 104–103 cfu/ml) in DAS-ELISA. Monoclonal antibody Mn-Cs1 showed a high level of specificity. It reacted neither with bacterial strains of closely related subspecies of Clavibacter michiganensis (C. m. subsp. michiganensis and C. m. subsp. insidiosus) nor with the saprophytic bacteria Pseudomonas fluorescens and Pantoea agglomerans.

scholarly journals Teknik Inokulasi Buatan Clavibacter michiganensis subsp. sepedonicus, Penyebab Penyakit Busuk Cincin Bakteri, pada Tanaman Kentang (Solanum tuberosum L.)

Agrikultura ◽  
2016 ◽  
Vol 27 (2) ◽  
Author(s):  
Luciana Djaya ◽  
Ineu Sulastrini ◽  
Iin Rusita
Keyword(s):  
Solanum Tuberosum ◽  
Solanum Tuberosum L ◽  
Block Design ◽  
Clavibacter Michiganensis ◽  
Bacterial Ring Rot ◽  
Seed Tubers ◽  
Clavibacter Michiganensis Subsp ◽  
Ring Rot ◽  
Inoculation Techniques ◽  
Rot Disease

ABSTRACT Inoculation Techniques of Clavibacter michiganensis subsp. sepedonicus, the Cause of Bacterial Ring Rot Disease, on Potato (Solanum tuberosum L.). Clavibacter michiganensis subsp. sepedonicus, the cause of bacterial ring rot disease on potatoes, has been detected in potato fields in Pangalengan. To anticipate the spread of the pathogen, researches on the desease epidemiology are urgent to be carried out. Artificial inoculation techniques will be useful in the epidemiological studies. The objective of this reasearch was to evaluate some inoculation techniques, which are simple, cheap and fast in causing disease symptoms. The experiment was carried out at the laboratory and glasshouse of Balai Penelitian Tanaman Sayuran (Balitsa), Lembang. The experiment was arranged in the randomized block design with five treatments of inoculation technique and five replications. The treatments were (a) soaking wounded seed tubers in pathogen suspension, (b) soaking not wounded seed tubers in pathogen suspension, (c) pathogen suspension was injected into leaf axil, (d) pathogen suspension was injected into seed tubers, and (e) pathogen suspension was poured into the planting holes. The results showed that stabbing and soaking tubers in pathogen suspension caused the shortest incubation period (17 days after inoculation) and the highest disease incidence (60%). Keywords : Clavibacter michiganensis subsp. sepedonicus, Bacterial ring rot, Potatoes, Inoculation techniquesABSTRAKClavibacter michiganensis subsp. sepedonicus (Cms), penyebab penyakit busuk cincin bakteri pada tanaman kentang, telah terdeteksi keberadaannya pada pertanaman kentang di Pangalengan. Dalam upaya mencegah penyebaran penyakit busuk cincin bakteri di Indonesia, perlu adanya studi epidemiologi patogen tersebut. Pada penelitian epidemiologi akan diperlukan cara menginokulasi tanaman secara buatan. Tujuan dari penelitian ini adalah untuk mendapatkan teknik inokulasi buatan yang dapat menyebabkan periode inkubasi yang lebih singkat dan persentase kejadian penyakit busuk cincin bakteri paling tinggi pada tanaman kentang. Percobaan dilaksanakan di rumah kaca dan laboratorium penyakit Balai Penelitian Tanaman Sayuran (Balitsa), Lembang. Percobaan dirancang secara rancangan acak kelompok dengan lima perlakuan teknik inokulasi Cms dan lima ulangan, yaitu inokulasi dengan (a) merendam ubi benih yang telah dilukai dalam suspensi bakteri, (b) merendam ubi benih tanpa dilukai dalam suspensi bakteri, (c) suspensi bakteri ditusukkan pada ketiak daun tanaman kentang, (d) suspensi bakteri ditusukkan pada ubi benih, dan (e) suspensi bakteri disiramkan pada lubang tanam. Masing-masing ulangan terdiri dari lima tanaman. Hasil penelitian menunjukkan bahwa dari lima teknik inokulasi buatan yang dilakukan, inokulasi dengan melukai ubi benih dan merendamnya dalam suspensi patogen menghasilkanJurnal Agrikultura 2016, 27 (2): 66-71ISSN 0853-2885Teknik Inokulasi Buatan Clavibacter….67periode inkubasi tersingkat, yaitu 17 hari setelah inokulasi, dan persentase kejadian penyakit tertinggi yaitu sebesar 60%.Kata Kunci : Clavibacter michiganensis subsp. sepedonicus, Busuk cincin bakteri, Kentang, Inokulasi buatan

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

1992 ◽  
Vol 72 (3) ◽  
pp. 943-953 ◽  
Author(s):  
B. A. Schuld ◽  
M. D. Harrison ◽  
J. Crane
Keyword(s):  
Solanum Tuberosum L ◽  
Solanum Melongena ◽  
Shoot Cultures ◽  
Clavibacter Michiganensis ◽  
Bacterial Ring Rot ◽  
Inoculum Level ◽  
Clavibacter Michiganensis Subsp ◽  
Nodal Cuttings ◽  
Symptomless Infection ◽  
Culturing Conditions

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.

scholarly journals Immunochemical, biochemical and pathogenic properties of fluidal and intermediate strains of Clavibacter michiganensis subsp. sepedonicus

2017 ◽  
Vol 38 (SI 2 - 6th Conf EFPP 2002) ◽  
pp. 291-293
Author(s):  
B. Kokošková ◽  
R. Jeřabková
Keyword(s):  
Monoclonal Antibodies ◽  
Metabolic Activity ◽  
Species Level ◽  
Colony Morphology ◽  
Clavibacter Michiganensis ◽  
Clavibacter Michiganensis Subsp ◽  
Ring Rot ◽  
Atypical Cells ◽  
Das Elisa

Clavibacter michiganensis subsp. sepedonicus (Cms), causing ring rot of potato, is a quarantine bacterium. According to colony morphology, Cms occurs mostly as fluidal (smooth), but sometime as intermediate (rough, i.e. less fluidal) variants, too. Commercial monoclonal antibodies (Agdia, USA) were used for determination of 40 Cms strains representing both forms. All Cms strains were reliably identified by IFAS, but atypical cells were sometime recorded in population of intermediate strains. The fluidal Cms strains were more reliably identified using DAS-ELISA and the Biolog GP MicroPlate System<sup>TM</sup> than intermediate strains. The intermediate Cms strains had decreased metabolic activity compared with fluidal strains and that is why they were identified only to the genera or to the species level or not identified. The differences among fluidal and intermediate Cms strains were recorded also in bioassay on eggplants. The intermediate Cms strains caused atypical or no symptoms with comparison to fluidal strains.

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

1994 ◽  
Vol 40 (12) ◽  
pp. 1007-1018 ◽  
Author(s):  
J. L. W. Rademaker ◽  
J. D. Janse
Keyword(s):  
Polymerase Chain Reaction ◽  
Polymerase Chain Reaction Product ◽  
Enzyme Analysis ◽  
Restriction Enzyme Analysis ◽  
Bacterial Strains ◽  
Clavibacter Michiganensis ◽  
Chain Reaction ◽  
Clavibacter Michiganensis Subsp ◽  
Detection And Identification ◽  
Polymerase Chain

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.

scholarly journals Clavibacter michiganensis subsp. Sepedonicus Elicits a Hypersensitive Response in Tobacco and Secretes Hypersensitive Response-Inducing Protein(s)

1997 ◽  
Vol 87 (7) ◽  
pp. 678-684 ◽  
Author(s):  
R. Nissinen ◽  
F.-M. Lai ◽  
M. J. Laine ◽  
P. J. Bauer ◽  
A. A. Reilley ◽  
...  
Keyword(s):  
Hypersensitive Response ◽  
Host Plants ◽  
Virulent Strain ◽  
Deae Cellulose ◽  
Protein S ◽  
Clavibacter Michiganensis ◽  
Bacterial Ring Rot ◽  
Clavibacter Michiganensis Subsp ◽  
Virulent Strains ◽  
Necrotic Response

Strains of Clavibacter michiganensis subsp. sepedonicus, causal agent of bacterial ring rot of potato, showed marked differences in virulence on host plants. When infiltrated into tobacco leaves, virulent strains caused a rapid localized necrotic response (within 24 to 48 h) characteristic of the hypersensitive response (HR), whereas nonpathogenic strains did not. Concentrated cell-free culture supernatants (CCS) from virulent strains caused a necrotic reaction on tobacco, whereas CCS from nonpathogenic strains did not. The necrosis-inducing activity was heat stable and protease sensitive. Inhibitors of eukaryotic metabolism suppressed the necrotic reaction of tobacco to CCS. No necrotic response was observed when host plants were infiltrated with either cells or CCS from virulent strains. HR-inducing protein(s) from a virulent strain separated from the majority of other proteins on DEAE cellulose at 250 to 300 mM NaCl. Ammonium sulfate-precipitated proteins from a virulent strain produced a necrotic reaction at a total protein concentration of 18 μg/ml, whereas those from a nonpathogenic strain did not, even at a concentration of 180 μg/ml. We conclude that virulent strains of C. michiganensis subsp. sepedonicus elicit a typical HR in tobacco and secrete proteinaceous elicitor(s) of the nonhost HR.

scholarly journals New Type of Antimicrobial Protein Produced by the Plant Pathogen Clavibacter michiganensis subsp. michiganensis

2013 ◽  
Vol 79 (18) ◽  
pp. 5721-5727 ◽  
Author(s):  
Zhanliang Liu ◽  
Ping Ma ◽  
Ingrid Holtsmark ◽  
Morten Skaugen ◽  
Vincent G. H. Eijsink ◽  
...  
Keyword(s):  
Antimicrobial Protein ◽  
Clavibacter Michiganensis ◽  
Bacterial Ring Rot ◽  
Content Type ◽  
Highly Active ◽  
Clavibacter Michiganensis Subsp ◽  
Ring Rot ◽  
Inhibitory Spectrum ◽  
New Type ◽  
Tomato Pathogen

ABSTRACTIt has previously been shown that the tomato pathogenClavibacter michiganensissubsp.michiganensissecretes a 14-kDa protein,C. michiganensissubsp.michiganensisAMP-I (CmmAMP-I), that inhibits growth ofClavibacter michiganensissubsp.sepedonicus, the causal agent of bacterial ring rot of potato. Using sequences obtained from tryptic fragments, we have identified the gene encodingCmmAMP-I and we have recombinantly produced the protein with an N-terminal intein tag. The gene sequence showed thatCmmAMP-I contains a typical N-terminal signal peptide for Sec-dependent secretion. The recombinant protein was highly active, with 50% growth inhibition (IC50) of approximately 10 pmol, but was not toxic to potato leaves or tubers.CmmAMP-I does not resemble any known protein and thus represents a completely new type of bacteriocin. Due to its high antimicrobial activity and its very narrow inhibitory spectrum,CmmAMP-1 may be of interest in combating potato ring rot disease.

scholarly journals Preparation and use of monoclonal antibodies  to detect Phytophthora nicotianae  var. nicotianae

1999 ◽  
Vol 35 (No. 2) ◽  
pp. 41-46
Author(s):  
B. Pekárová-Kyněrová ◽  
M. Kutíková
Keyword(s):  
Monoclonal Antibody ◽  
Monoclonal Antibodies ◽  
Fusarium Oxysporum ◽  
Indirect Elisa ◽  
Phytophthora Nicotianae ◽  
Clavibacter Michiganensis ◽  
Tomato Plants ◽  
Clavibacter Michiganensis Subsp

A monoclonal antibody (MAb 18) was prepared against purified mycelial proteins from Phytophthora nicotianae var. nicotianae. The specificity of MAb 18 (lgG class) was tested using indirect ELISA (PTA-ELISA).It cross-reacted with Phytophthora cacto­ rum, P. cinrzamomi, P. cryptogea, P. fragariae) but not with other fungi (Fusarium  oxysporum, Pythium ultimwn and P. oligan­ drwn) and bacteria (Clavibacter michiganensis subsp. michiganensis) isolated from tomato. Phytophthora nicotianae var. nicotianae was detected in roots and basal stems of artificially infected young tomato plants using indirect ELISA and immunoprinting.

scholarly journals Resistance to Bacterial Canker in Tomato (Lycopersicon hirsutum LA407) and its Progeny Derived from Crosses to L. esculentum

Plant Disease ◽  
2001 ◽  
Vol 85 (11) ◽  
pp. 1171-1176 ◽  
Author(s):  
David M. Francis ◽  
Eileen Kabelka ◽  
Julia Bell ◽  
Barb Franchino ◽  
Dina St. Clair
Keyword(s):  
Correlation Coefficients ◽  
Field Trials ◽  
Dna Fingerprint ◽  
Bacterial Canker ◽  
Lycopersicon Hirsutum ◽  
Bacterial Strains ◽  
Clavibacter Michiganensis ◽  
Clavibacter Michiganensis Subsp ◽  
Significant Yield ◽  
Linear Correlations

Bacterial canker caused by Clavibacter michiganensis subsp. michiganensis causes significant yield losses on tomatoes grown in a humid environment. This study was conducted to identify a source of resistance that could be easily crossed to cultivated tomato and to study the inheritance of resistance. Diverse bacterial strains representative of the major DNA fingerprint classes endemic to North America were used to screen germ plasm and populations derived from wide crosses. Partial resistance to genetically characterized and distinct strains of C. michiganensis subsp. michiganensis was identified in a wild relative of cultivated tomato, Lycopersicon hirsutum Lycopersicon accession (LA)407. The level of resistance in LA407 was not significantly different from that of the resistant L. peruvianum control, LA2157. Resistance from LA407 was recovered in lines from a BC2S4 inbred backcross (IBC) population in both greenhouse and field trials. Linear correlations between field and greenhouse resistance scores were significant, though correlation coefficients tended to be low. Variance components for genetic and environmental variation in resistance were used to estimate broad-sense heritability in the IBC population. These estimates were moderate to high, ranging from 0.34 to 0.85. The number of genes contributing to resistance was estimated from four trials, with most estimates falling in the range of one to three loci. Two lines from the IBC population, IBL 2353 and IBL 2361, were identified as sources that retain resistance in a genetic background that has a theoretical L. esculentum genome content of 87.5%.

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