scholarly journals Comparison of Dig-Labeled PCR, Nested PCR, and ELISA for the Detection of Clavibacter michiganensis subsp. sepedonicus in Field-Grown Potatoes

Plant Disease ◽  
2001 ◽  
Vol 85 (3) ◽  
pp. 261-266 ◽  
Author(s):  
I.-M. Lee ◽  
L. A. Lukaesko ◽  
C. J. M. Maroon
Keyword(s):  
Nested Pcr ◽  
Colorimetric Assay ◽  
Detection Sensitivity ◽  
Enzyme Linked Immunosorbent Assay ◽  
Clavibacter Michiganensis ◽  
Insertion Element ◽  
Nylon Membrane ◽  
Clavibacter Michiganensis Subsp ◽  
Ring Rot ◽  
Pcr Products

The sensitivity of digoxigenin- (dig-) labeled polymerase chain reaction (PCR) was compared with nested PCR and enzyme-linked immunosorbent assay (ELISA) for the detection of the potato ring rot bacterium, Clavibacter michiganensis subsp. sepedonicus, in seed potatoes and stem tissues. The bacterial DNA was extracted from chopped tuber or stem tissue by a modified hot alkaline DNA extraction method. C. michiganensis subsp. sepedonicus specific DNA sequence was amplified by dig-labeled PCR with the primer pair CMSIF1/CMSIR1 previously designed based on the insertion element IS1121 of C. michiganensis subsp. sepedonicus. Dig-labeled PCR products were then blotted on a nylon membrane and the signal was detected by a colorimetric assay using alkaline phosphatase. The new assay procedure has a detection sensitivity close to that of nested PCR and simplicity of the standard ELISA procedure commonly used commercially. The dig-labeled PCR assay was more sensitive than ELISA and can be used to detect C. michiganensis subsp. sepedonicus in symptomless field potato tubers.

scholarly journals A Real-Time PCR Assay for the Detection of Clavibacter michiganensis subsp. sepedonicus Based on the Cellulase A Gene Sequence

Plant Disease ◽  
2009 ◽  
Vol 93 (6) ◽  
pp. 649-659 ◽  
Author(s):  
Neil C. Gudmestad ◽  
Ipsita Mallik ◽  
Julie S. Pasche ◽  
Nolan R. Anderson ◽  
Kasia Kinzer
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Gene Sequence ◽  
Certified Seed ◽  
Pcr Primers ◽  
Enzyme Linked Immunosorbent Assay ◽  
Clavibacter Michiganensis ◽  
Pcr Assay ◽  
Clavibacter Michiganensis Subsp ◽  
Ring Rot

Clavibacter michiganensis subsp. sepedonicus, causal agent of bacterial ring rot (BRR) of potato (Solanum tuberosum), is a globally important quarantine pathogen that is managed in North America using zero tolerance regulations in the certified seed industry. C. michiganensis subsp. sepedonicus is well documented to cause symptomless infections in potato, contributing to its persistence in certified seed stocks. Reliable laboratory methods to detect symptomless infections with a high degree of sensitivity could assist in the reduction of inoculum in certified seed potato stocks. A real-time polymerase chain reaction (PCR) assay was developed using the cellulase A (CelA) gene sequence as the basis for primer design. CelA primers were specific to C. michiganensis subsp. sepedonicus grown in vitro and did not detect any other coryneform bacteria or potato pathogenic bacteria but did detect 69 strains of C. michiganensis subsp. sepedonicus. The CelA real-time PCR assay was more sensitive than immunofluorescence (IFA) and Cms50/72a PCR assays in detecting C. michiganensis subsp. sepedonicus in infected potato tuber cores blended with healthy tuber cores in simulated seed lot contamination experiments. CelA primers detected nonmucoid and mucoid strains with equivalent sensitivity. In naturally infected seed lots, CelA PCR primers also were more sensitive in detecting symptomless infections of C. michiganensis subsp. sepedonicus in seed tubers prior to planting compared to Cms50/72a PCR primers, IFA, and enzyme-linked immunosorbent assay. A real-time PCR format using the newly developed CelA primers proved to be a very robust detection tool for C. michiganensis subsp. sepedonicus with the added advantage of detecting only virulent strains of the ring rot bacterium.

scholarly journals Bacterial Ring Rot of Potatoes Caused by Clavibacter michiganensis subsp. sepedonicus in Lithuania

Plant Disease ◽  
2001 ◽  
Vol 85 (10) ◽  
pp. 1119-1119
Author(s):  
L. Baranauskaite ◽  
M. Vasinauskiene
Keyword(s):  
Plant Protection ◽  
Polyclonal Antibodies ◽  
Solanum Tuberosum L ◽  
Disease Incidence ◽  
Potato Production ◽  
Enzyme Linked Immunosorbent Assay ◽  
Clavibacter Michiganensis ◽  
Bacterial Ring Rot ◽  
Clavibacter Michiganensis Subsp ◽  
Ring Rot

Bacterial ring rot of potato (Solanum tuberosum L.) was observed for the first time in Lithuania from 1998 to 1999. The disease, caused by Clavibacter michiganensis subsp. sepedonicus, is considered one of the most important bacterial diseases of potato. Nearly all countries that produce potatoes report the presence of the bacterium (1). C. michiganensis subsp. sepedonicus is listed as an A2 quarantine pathogen by the European and Mediterranean Plant Protection Organization. The pathogen has a latent period that can last for several generations of potato production, thus increasing the risk for further dissemination of the disease. Plant wilting and tuber rotting, characteristic symptoms of the disease, were observed in various localities in Lithuania. The disease was found in 12 potato varieties. Disease incidence approached 5% in the field and 5 to 21% in storage. The presence of C. michiganensis subsp. sepedonicus in diseased as well as asymptomatic plant tissue was determined by immunofluorescence cell staining and enzyme-linked immunosorbent assay with the use of monoclonal and polyclonal antibodies, respectively. More than 350 potato samples were tested. Twenty-five samples tested positive by these methods. Pathogenicity tests of 30 samples were conducted on eggplant. Characteristic symptoms were found on 21 plants. The bacteria were reisolated on nutrient dextrose agar medium from 12 inoculated eggplants. For confirmation of six suspicious strains, polymerase chain reaction with a specific Cms50 primer set was employed (2). In three cases, positive results were obtained. References: (1) I. M. Smith and L. M. F. Charles, eds. 1998. Distribution of Maps of Quarantine Pests for Europe. CAB International, Wallingford, U.K. (2) D. Mills et al. Phytopathology 87:853, 1997.

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

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.

scholarly journals Wilt Pathogens of Solanaceae in Tanzania: Clavibacter michiganensis subsp. michiganensis, Pseudomonas corrugata, and Ralstonia solanacearum

Plant Disease ◽  
1999 ◽  
Vol 83 (11) ◽  
pp. 1070-1070 ◽  
Author(s):  
R. Black ◽  
S. Seal ◽  
Z. Abubakar ◽  
R. Nono-Womdim ◽  
I. Swai
Keyword(s):  
Bacterial Wilt ◽  
Ralstonia Solanacearum ◽  
Polymyxin B ◽  
Enzyme Linked Immunosorbent Assay ◽  
Bacterial Canker ◽  
Clavibacter Michiganensis ◽  
Pseudomonas Corrugata ◽  
Tomato Seed ◽  
Clavibacter Michiganensis Subsp ◽  
Northern Highlands

Surveys of vegetables in the southern and northern growing regions of mainland Tanzania and Zanzibar during 1997 and 1998 indicated the presence of three wilt pathogens of tomato. Clavibacter michiganensis subsp. michiganensis (cause of bacterial canker) was isolated from wilting plants in the southern highlands and Lushoto District (Tanga Region, north) on selective King's medium B with polymyxin B (1). The identity of the isolates was confirmed by cultural, morphological, and biochemical characteristics and a plate-trapped antigen enzyme-linked immunosorbent assay (PTA-ELISA) kit (Pathoset 113-08, Adgen, Auchincruive, U.K.). Pathogenicity was confirmed by host inoculation. In addition, the bacterium was detected directly by the same methods in several sources of seed, including commercial farmers' saved seed and seed extracted from infected plants. Of 61 tomato seed lots tested, 18 samples were positive. Ralstonia solanacearum (cause of bacterial wilt) was isolated from tomato and potato by semiselective media and detected directly in stem and tuber tissues, respectively, by polymerase chain reaction and ELISA (4) in all vegetable-growing areas surveyed. In 1998, R. solanacearum was detected for the first time in Zanzibar on tomato and eggplant. Pathogenicity was confirmed by inoculation on and reisolation from tomato seedlings of cv. Money Maker. Only biovar 3 (2) occurred in tomato. Biovar 3 also was found in midaltitude potato. Biovar 2 has been found only in potato plants grown above 1,500 m. Pseudomonas corrugata (cause of pith necrosis) was isolated from tomato on semiselective media at only one location. From wilted tomato plants in the southern highlands, 38% of samples tested positive for C. michiganensis subsp. michiganensis and ≈ 10% for R. solanacearum. Of samples collected from the northern highlands, 43% tested positive for R. solanacearum. Wilt incidence of ≈35% was observed in tomato fields where the bacterial wilt pathogen was isolated in the northern highlands compared with gt;90% incidence and almost total crop loss in tomato fields of the southern highlands infected with bacterial canker. Although all three pathogens caused systemic wilt of plants, bacterial canker occasionally caused downward turning of lower leaves, unilateral wilting, and marginal necrosis of leaflets as well as fruit spotting. In general, wilts caused by C. michiganensis subsp. michiganensis, R. solanacearum, and P. corrugata were not readily differentiated in Tanzania prior to this research. Plants with pith browning had often been assumed to be infected by P. corrugata, until R. solanacearum was isolated (3) frequently from such plants. References: (1) Anonymous. OEPP/EPPO Bull. 22:219, 1992. (2) A. C. Hayward. J. Appl. Bacteriol. 27:265, 1964. (3) J. B. Jones et al., eds. 1991. Compendium of Tomato Diseases. The American Phytopathological Society, St. Paul, MN. (4) S. Seal and J. G. Elphinstone. Pages 35–57 in: Bacterial Wilt. The Disease and Its Causative Agent, Pseudomonas solanacearum. A. C. Hayward and G. L. Hartman, eds. CAB International, Wallingford, U.K. 1994.

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 A Highly Efficient Transposon Mutagenesis System for the Tomato Pathogen Clavibacter michiganensis subsp. michiganensis

2001 ◽  
Vol 14 (11) ◽  
pp. 1312-1318 ◽  
Author(s):  
Oliver Kirchner ◽  
Karl-Heinz Gartemann ◽  
Eva-Maria Zellermann ◽  
Rudolf Eichenlaub ◽  
Annette Burger
Keyword(s):  
Transposon Mutagenesis ◽  
Saturation Mutagenesis ◽  
Clavibacter Michiganensis ◽  
Insertion Element ◽  
Suicide Vector ◽  
Clavibacter Michiganensis Subsp ◽  
Transposon Mutants ◽  
Insertion Sites ◽  
Tomato Pathogen ◽  
Electroporation Efficiency

A transposon mutagenesis system for Clavibacter michiganensis subsp. michiganensis was developed based on antibiotic resistance transposons that were derived from the insertion element IS1409 from Arthrobacter sp. strain TM1 NCIB12013. As a prerequisite, the electroporation efficiency was optimized by using unmethylated DNA and treatment of the cells with glycine such that about 5 × 106 transformants per μg of DNA were generally obtained. Electroporation of C. michiganensis subsp. michiganensis with a suicide vector carrying transposon Tn1409C resulted in approximately 1 × 103 transposon mutants per μg of DNA and thus is suitable for saturation mutagenesis. Analysis of Tn1409C insertion sites suggests a random mode of transposition. Transposition of Tn1409C was also demonstrated for other subspecies of C. michiganensis.

scholarly journals Bacterial Canker (Clavibacter michiganensis subsp. michiganensis) of Tomato in Commercial Seed Produced in Indonesia

Plant Disease ◽  
2004 ◽  
Vol 88 (6) ◽  
pp. 680-680 ◽  
Author(s):  
A. Anwar ◽  
P. S. van der Zouwen ◽  
S. Ilyas ◽  
J. M. van der Wolf
Keyword(s):  
Enzyme Linked Immunosorbent Assay ◽  
Pathogenicity Test ◽  
Bacterial Canker ◽  
Vegetable Crops ◽  
Clavibacter Michiganensis ◽  
Tomato Seed ◽  
Causal Organism ◽  
Tomato Production ◽  
Clavibacter Michiganensis Subsp ◽  
Bacterial Canker Of Tomato

In 2002, Clavibacter michiganensis subsp. michiganensis (Smith) Davis, the causal organism of bacterial canker of tomato (Lycopersicon esculentum), was isolated from two of six commercial asymptomatic tomato seed lots produced on Java in Indonesia. C. michiganensis subsp. michiganensis has not been reported in Indonesia previously. Methods based on the protocol of the International Seed Health Initiative were used to extract and identify the presence of C. michiganensis subsp. michiganensis in tomato seed. C. michiganensis subsp. michiganensis was isolated with dilution plating on the semiselective media D2ANX and mSCM. The identity of the colonies was confirmed by immunofluorescence microscopy, polymerase chain reaction (2), fatty methyl ester analysis, enzyme-linked immunosorbent assay based on monoclonal antibody 103 (1), and a pathogenicity test in which three replicate tomato plants were stem inoculated with 108 cells ml-1. Within 2 weeks, stripes on stems developed that split and exposed reddish brown cavities (stem cankers). The presence of C. michiganensis subsp. michiganensis poses a direct threat on tomato production, which is one of five economically most important vegetable crops in Indonesia. References: (1) A. Alvarez et al. Phytopathology 83:1405, 1993. (2) M. S. Santos et al. Seed Sci. Technol. 25:581, 1997.

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