scholarly journals Detection of Clavibacter michiganensis subsp. sepedonicus in Potato Tubers by BIO-PCR and an Automated Real-Time Fluorescence Detection System

Plant Disease ◽  
1999 ◽  
Vol 83 (12) ◽  
pp. 1095-1100 ◽  
Author(s):  
N. W. Schaad ◽  
Y. Berthier-Schaad ◽  
A. Sechler ◽  
D. Knorr
Keyword(s):  
Real Time ◽  
Detection System ◽  
Pcr Primers ◽  
Competitive Growth ◽  
Potato Tubers ◽  
Clavibacter Michiganensis ◽  
Clavibacter Michiganensis Subsp ◽  
Ring Rot ◽  
Large Numbers ◽  
Fluorescence Detection System

Ring rot of potato, caused by Clavibacter michiganensis subsp. sepedonicus, is one of the most regulated diseases of potatoes world wide. The organism is often difficult to detect in symptomless tubers because of low populations and slow competitive growth on available media. Polymerase chain reaction (PCR) primers and a fluorescent probe for use in the Perkin Elmer 7700 automated real time PCR detection system (TaqMan) were designed from a C. michiganensis subsp. sepedonicus-specific genomic DNA fragment for development of a BIO-PCR assay for C. michiganensis subsp. sepedonicus in potato tubers. Results of screening the primers with strains of C. michiganensis subsp. sepedonicus and other bacteria showed the primers to be specific. A total of 30 naturally infected ring rot suspect tubers were sampled by the core extract, shaker incubation procedure and assayed by (i) plating aliquots onto agar media, (ii) classical PCR, and (iii) BIO-PCR. In all, 4 tubers were positive by agar plating and pathogenicity tests, 8 by classical TaqMan PCR, and 26 by TaqMan BIO-PCR. We conclude that BIO-PCR combined with the TaqMan automated closed detection system is a rapid, reliable method of assaying large numbers of potato tuber extracts for C. michiganensis subsp. sepedonicus. Furthermore, for a large central laboratory running large numbers of PCR assays, the high-throughput TaqMan system can reduce costs per sample over the more labor-intensive classical PCR.

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.

Clavibacter michiganensis subsp. sepedonicus (potato ring rot).

2021 ◽  
Author(s):  
Solke de Boer
Keyword(s):  
Potato Production ◽  
Potato Tubers ◽  
Clavibacter Michiganensis ◽  
Clavibacter Michiganensis Subsp ◽  
Ring Rot ◽  
Cool Environment ◽  
Latently Infected ◽  
Important Means ◽  
Potato Ring Rot ◽  
On Farm

Abstract Clavibacter michiganensis subsp. sepedonicus has the propensity to exist asymptomatically as latent infections in potato (Solanum tuberosum); it is not known to naturally infect other plant species. Inadvertent dissemination of the bacterium to new places of production occurs with the movement of latently infected seed tubers used for planting. The bacterium also spreads from infected tubers through direct contact and by contamination of equipment used for potato production such as seed cutters, planters, harvesters, transport vehicles, storages, etc. C. michiganensis subp. sepedonicus survives for extended periods of many months to years in a dry and cool environment. Hence its persistence on farm equipment, in storages, and on transport vehicles is an important means by which the bacterium is maintained and spread within farm units and disseminated to other production units. It persists in the field in unharvested potato tubers (i.e. volunteers or ground keepers) and in infected potato plant debris.

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 An Internal Reaction Control for Routine Detection of Clavibacter michiganensis subsp. sepedonicus Using a Real-Time TaqMan PCR-Based Assay

Plant Disease ◽  
2008 ◽  
Vol 92 (5) ◽  
pp. 684-693 ◽  
Author(s):  
Donna S. Smith ◽  
Solke H. De Boer ◽  
Jane Gourley
Keyword(s):  
Real Time ◽  
Limit Of Detection ◽  
Taqman Assay ◽  
Clavibacter Michiganensis ◽  
Causal Organism ◽  
Melt Temperature ◽  
Clavibacter Michiganensis Subsp ◽  
Internal Reaction ◽  
Control Plasmid ◽  
Taqman Pcr

An internal reaction control was integrated into a TaqMan polymerase chain reaction (PCR) assay for the detection of Clavibacter michiganensis subsp. sepedonicus, the causal organism of bacterial ring rot of potato. The reaction control, cloned into plasmid pCmsC4, consisted of a sequence unrelated to C. michiganensis subsp. sepedonicus flanked by the primer sequences used in the TaqMan PCR, thus eliminating the need for multiplexing. Inclusion of the reaction control plasmid in the TaqMan assay had no effect on either the limit of detection or the specificity of the method. Addition of SYBR Green permitted melt analysis of PCR products. The 242-bp reaction control amplicon, with a melt temperature of approximately 94.5°C, could easily be distinguished from the 152-bp primary diagnostic target amplicon, which had a melt temperature of about 85.5°C. Electrophoretic analysis showed that appearance of either melt peak correlated well with the presence of the appropriate amplicon. Two different substances, guanidine-HCl and humic acid, inhibited the amplification of the reaction control at concentrations lower than those that inhibited the primary diagnostic target, demonstrating the reaction control's effectiveness in detecting inhibition or reaction failure. Using the reaction control plasmid, a quantitative threshold for inhibitor detection was established. This permitted the validation of negative results, and thus facilitated the use of TaqMan real-time PCR in the routine testing of diagnostic samples for C. michiganensis subsp. sepedonicus.

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