Persistence and Latency of Clavibacter michiganensis subsp. sepedonicus in Field-Grown Seed Potatoes

Whole seed tubers were inoculated with different inoculum doses of Clavibacter michiganensis subsp. sepedonicus and planted in the field. Ring rot symptom development and the persistence of C. michiganensis subsp. sepedonicus, in the absence of symptoms, was determined for four generations following introduction of inoculum. Seed tubers were not cut during propagation to preclude redistribution of inoculum. Detection of C. michiganensis subsp. sepedonicus was positively related to inoculum dose (r = 0.97) and rapidly declined with increasing generations. Although the frequency of detection was much less following inoculation with ≤102 cells versus inoculation with ≥104 cells, all inoculum doses established latent infections that were undetected for at least one, and up to three, generations. Latency for three generations, characterized by C. michiganensis subsp. sepedonicus detection or ring rot symptom development during the fourth generation, was the longest time interval tested. The occurrence of latent C. michiganensis subsp. sepedonicus infections and the persistence of inadvertent inoculum sources may, at least partially, explain our inability to eradicate ring rot based on visible symptom expression.

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

Agrikultura ◽

10.24198/agrikultura.v27i2.9967 ◽

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

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

Plant Protection Science ◽

10.17221/10471-pps ◽

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.

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Nested PCR for ultrasensitive detection of the potato ring rot bacterium, Clavibacter michiganensis subsp. sepedonicus.

Applied and Environmental Microbiology ◽

10.1128/aem.63.7.2625-2630.1997 ◽

1997 ◽

Vol 63 (7) ◽

pp. 2625-2630 ◽

Cited By ~ 29

Author(s):

I M Lee ◽

I M Bartoszyk ◽

D E Gundersen ◽

B Mogen ◽

R E Davis

Keyword(s):

Nested Pcr ◽

Ultrasensitive Detection ◽

Clavibacter Michiganensis ◽

Clavibacter Michiganensis Subsp ◽

Ring Rot ◽

Potato Ring Rot

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

Plant Disease ◽

10.1094/pdis-93-6-0649 ◽

2009 ◽

Vol 93 (6) ◽

pp. 649-659 ◽

Cited By ~ 16

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.

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

Applied and Environmental Microbiology ◽

10.1128/aem.01065-13 ◽

2013 ◽

Vol 79 (18) ◽

pp. 5721-5727 ◽

Cited By ~ 10

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.

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

Plant Disease ◽

10.1094/pdis.1999.83.12.1095 ◽

1999 ◽

Vol 83 (12) ◽

pp. 1095-1100 ◽

Cited By ~ 102

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.

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Lateral flow immunoassay for rapid detection of potato ring rot caused by Clavibacter michiganensis subsp. sepedonicus

Applied Biochemistry and Microbiology ◽

10.1134/s0003683814120011 ◽

2014 ◽

Vol 50 (6) ◽

pp. 675-682 ◽

Cited By ~ 6

Author(s):

I. V. Safenkova ◽

I. A. Zaitsev ◽

G. K. Pankratova ◽

Yu. A. Varitsev ◽

A. V. Zherdev ◽

...

Keyword(s):

Rapid Detection ◽

Lateral Flow ◽

Lateral Flow Immunoassay ◽

Clavibacter Michiganensis ◽

Clavibacter Michiganensis Subsp ◽

Ring Rot ◽

Potato Ring Rot

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

Canadian Journal of Microbiology ◽

10.1139/m94-024 ◽

1994 ◽

Vol 40 (2) ◽

pp. 148-151 ◽

Cited By ~ 18

Author(s):

Giuseppe Firrao ◽

Romano Locci

Keyword(s):

Polymerase Chain Reaction ◽

Clavibacter Michiganensis ◽

Chain Reaction ◽

Chain Reactions ◽

Polymerase Chain Reactions ◽

Clavibacter Michiganensis Subsp ◽

Ring Rot ◽

Dna Fragment ◽

Polymerase Chain ◽

Polymerase Chain Reaction Primers

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. michiganensis 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 × 103 bacteria.Key words: detection, potato ring rot, Corynebacterium sepedonicum.

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