Biological suppression of potato ring rot by fluorescent pseudomonads.

Three strains of fluorescent pseudomonads (IS-1, IS-2, and IS-3) isolated from potato underground stems with roots showed in vitro antibiosis against 30 strains of the ring rot bacterium Clavibacter michiganensis subsp. sepedonicus. On the basis of morphological and biochemical tests and fatty acid analysis, IS-1 and IS-2 were identified as Pseudomonas aureofaciens and IS-3 was identified as P. fluorescens biovar III. IS-1 was the most inhibitory to C. michiganensis subsp. sepedonicus strains in vitro, followed by IS-3 and IS-2. Suppression of ring rot by these antagonists was demonstrated in greenhouse trials with stem-cultured potato (cv. Russet Burbank) seedlings. Although each antagonist significantly reduced C. michiganensis subsp. sepedonicus populations, only IS-1 reduced infection by C. michiganensis subsp. sepedonicus. In a second experiment, treatment with IS-1 (10(9) CFU/ml) significantly reduced ring rot infection by 23.4 to 26.7% after 5 to 8 weeks. The average C. michiganensis subsp. sepedonicus population was also significantly reduced by 50 to 52%. Application of different combinations of antagonist strains was not more effective than single-strain treatment.     

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

Oligonucleotide primers derived from sequences of the 16S rRNA gene (CMR16F1, CMR16R1, CMR16F2, and CMR16R2) and insertion element IS1121 of Clavibacter michiganensis subsp. sepedonicus (CMSIF1, CMSIR1, CMSIF2, and CMISR2) were used in nested PCR to detect the potato ring rot bacterium C. michiganensis subsp. sepedonicus. Nested PCR with primer pair CMSIF1-CMSIR1 followed by primer pair CMSIF2-CMSIR2 specifically detected C. michiganensis subsp. sepedonicus, while nested PCR with CMR16F1-CMR16R1 followed by CMR16F2-CMR16R2 detected C. michiganensis subsp. sepedonicus and the other C. michiganensis subspecies. In the latter case, C. michiganensis subsp. sepedonicus can be differentiated from the other subspecies by restriction fragment length polymorphism (RFLP) analyses of the nested PCR products (16S rDNA sequences). The nested PCR assays developed in this work allow ultrasensitive detection of very low titers of C. michiganensis subsp. sepedonicus which may be present in symptomiess potato plants or tubers and which cannot be readily detected by direct PCR (single PCR amplification). RFLP analysis of PCR products provides for an unambiguous confirmation of the identify of C. michiganensis subsp. sepedonicus.     

Stable transformation of the gram-positive phytopathogenic bacterium Clavibacter michiganensis subsp. sepedonicus with several cloning vectors.

In this paper we describe transformation of Clavibacter michiganensis subsp. sepedonicus, the potato ring rot bacterium, with plasmid vectors. Three of the plasmids used, pDM100, pDM302, and pDM306, contain the origin of replication from pCM1, a native plasmid of C. michiganensis subsp. michiganensis. We constructed two new cloning vectors, pHN205 and pHN216, by using the origin of replication of pCM2, another native plasmid of C. michiganensis subsp. michiganensis. Plasmids pDM302, pHN205, and pHN216 were stably maintained without antibiotic selection in various strains of C. michiganensis subsp. sepedonicus. We observed that for a single plasmid, different strains of C. michiganensis subsp. sepedonicus showed significantly different transformation efficiencies. We also found unexplained strain-to-strain differences in stability with various plasmid constructions containing different arrangements of antibiotic resistance genes and origins of replication. We examined the effect of a number of factors on transformation efficiency. The best transformation efficiencies were obtained when C. michiganensis subsp. sepedonicus cells were grown on DM agar plates, harvested during the early exponential growth phase, and used fresh (without freezing) for electroporation. The maximal transformation efficiency obtained was 4.6 x 10(4) CFU/microgram of pHN216 plasmid DNA. To demonstrate the utility of this transformation system, we cloned a beta-1,4-endoglucanase-encoding gene from C. michiganensis subsp. sepedonicus into pHN216. When this construction, pHN216:C8, was electroporated into competent cells of a cellulase-deficient mutant, it restored cellulase production to almost wild-type levels.     

Purification, characterization, and gene sequence of michiganin A, an actagardine-like lantibiotic produced by the tomato pathogen Clavibacter michiganensis subsp. michiganensis

Members of the actinomycete genus Clavibacter are known to produce antimicrobial compounds, but so far none of these compounds has been purified and characterized. We have isolated an antimicrobial peptide, michiganin A, from the tomato pathogen Clavibacter michiganensis subsp. michiganensis, using ammonium sulfate precipitation followed by cation-exchange and reversed-phase chromatography steps. Upon chemical derivatization of putative dehydrated amino acids and lanthionine bridges by alkaline ethanethiol, Edman degradation yielded sequence information that proved to be sufficient for cloning of the gene by a genome-walking strategy. The mature unmodified peptide consists of 21 amino acids, SSSGWLCTLTIECGTIICACR. All of the threonine residues undergo dehydration, and three of them interact with cysteines via thioether bonds to form methyllanthionine bridges. Michiganin A resembles actagardine, a type B lantibiotic with a known three-dimensional structure, produced by Actinoplanes liguriae, which is a filamentous actinomycete. The DNA sequence of the gene showed that the michiganin A precursor contains an unusual putative signal peptide with no similarity to well-known secretion signals and only very limited similarity to the (only two) available leader peptides of other type B lantibiotics. Michiganin A inhibits the growth of Clavibacter michiganensis subsp. sepedonicus, the causal agent of ring rot of potatoes, with MICs in the low nanomolar range. Thus, michiganin A may have some potential in biological control of potato ring rot.     

The tomato pathogen Clavibacter michiganensis ssp. michiganensis: producer of several antimicrobial substances

AIM: To purify and analyse antimicrobial substances produced by the tomato pathogen Clavibacter michiganensis ssp. michiganensis (Cmm), with potential application in control of Clavibacter michiganensis ssp. sepedonicus (Cms), the causal agent of bacterial ring rot of potato. METHODS AND RESULTS: After selection of a suitable producer and indicator strain, antimicrobial compounds were isolated using chromatographic techniques. The resulting preparations were analysed with respect to heat and protease sensitivity, amino acid composition, amino acid sequence and mass. Using this procedure we discovered one post-translationally modified 2145 Da peptide bacteriocin, one 14 kDa antimicrobial protein as well as low molecular weight (<1000 Da) antimicrobial compounds, putatively belonging to the tunicamycin family. CONCLUSIONS: Clavibacter michiganensis ssp. michiganensis produces various antibacterial substances that are active against Cms. SIGNIFICANCE AND IMPACT OF THE STUDY: This study describes the first attempt to characterize antimicrobial substances from Cmm at the molecular level. This is an important step towards investigation of the possible use of these compounds to control the potato ring rot pathogen.     

Linear plasmid in the genome of Clavibacter michiganensis subsp. sepedonicus

Contour-clamped homogeneous electric field gel analysis of genomic DNA of the plant pathogen Clavibacter michiganensis subsp. sepedonicus revealed the presence of a previously unreported extrachromosomal element. This new element was demonstrated to be a linear plasmid. Of 11 strains evaluated, all contained either a 90-kb (pCSL1) or a 140-kb (pCSL2) linear plasmid.     

Determination of genetic differences between fluid and nonfluid variants of<Emphasis Type="Italic">Clavibacter michiganensis</Emphasis> subsp.<Emphasis Type="Italic">sepedonicus</Emphasis> using rep-PCR technique

Testing of 23 isolates of Clavibacter michiganensis subsp. sepedonicus for analysis by rep-PCR (using BOX, ERIC, REP primer sets) was used for the purpose of localization of genetic markers for fluid and/or nonfluid strains. None of the primer sets was successful in detecting genetic differences between the isolates and no polymorphism was generated.     

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

A lateral flow immunoassay for the rapid detection of Clavibacter michiganensis subsp. sepedonicus bacteria causing potato ring rot was developed. Multimembrane composites (test strips) containing polyclonal antibodies against the bacteria and gold nanoparticle-antibody conjugates were used for the analysis. The test strips are suitable for the analysis of potato tuber and leaf extracts within 10 min; the detection limit of bacteria is 4 × 10⁵ cells/mL. No cross-reactivity with strains of Clavibacter michiganensis subsp. michiganensis, Pectobacterium carotovorum subsp. carotovorum and saprophytes of healthy potato plants was detected. The results of analysis of 26 potato samples by the developed tests were compared with those obtained by the PCR method and using the commercial enzyme immunoassay kits. The results of lateral flow immunoassay were confirmed in 96.2% of cases, which supports the high correlation with other analytical approaches. The developed immunoassay may be considered as a promising means of phytosanitary control.     

The Probability of Detecting Clavibacter michiganensis subsp. sepedonicus by Indexing Seed Potato Lots with Serological Tests

Detection of the bacterial ring pot pathogen ( Clavibacter michiganensis subsp. spedonicus ) in seed potato lots by laboratory indexing complements visual inspection. The probability of detecting symptomless infections is a function of sample size and incidence of infection. We determined the incidence of asymptomatic stem and tuber infections in four potato cultivars at three levels of inoculum. At the high inoculum level, 51–93% of stems were infected at 80 days after planting, and 10–59% of the tubers were infected at harvest. The effect of the different percentages of infected stems and tubers on the probability of detection for simple random sampling was calculated for a constant sample size. The actual detection levels for two cultivars planted in field plots with predetermined incidence levels of ring rot infected plants were reasonably close to predicted probabilities.     

Clavibacter michiganensis subsp. michiganensis: first steps in the understanding of virulence of a Gram-positive phytopathogenic bacterium

Clavibacter michiganensis subsp. michiganensis is a plant-pathogenic actinomycete. It infects tomato, spreads through the xylem and causes bacterial wilt and canker. The wild-type strain NCPPB382 carries two plasmids, pCM1 and pCM2. The cured plasmid-free derivative CMM100 is still able to colonize tomato, but no disease symptoms develop indicating that all genes required for successful infection, establishment and growth in the plant reside on the chromosome. Both plasmids carry one virulence factor, a gene encoding a cellulase, CelA in case of pCM1 and a putative serine protease Pat-1 on pCM2. These genes can independently convert the non-virulent strain CMM100 into a pathogen causing wilt on tomatoes. Currently, genome projects for Cmm and the closely related potato-pathogen C. michiganensis subsp. sepedonicus have been initiated. The data from the genome project shall give clues on further genes involved in plant-microbe interaction that can be tested experimentally. Especially, identification of genes related to host-specificity through genome comparison of the two subspecies might be possible.     

Detection of Clavibacter michiganensis subsp. sepedonicus by AmpliDet RNA,a new technology based on real time monitoring of NASBA amplicons with a molecular beacon

AIMS: To develop a procedure for direct detection of viable cells of Clavibacter michiganensis subsp. sepedonicus (Cms), the causal organism of bacterial ring rot in potato, based on AmpliDet RNA, in which amplicons generated by nucleic acid sequence based amplification (NASBA) are monitored in real time with a molecular beacon. METHODS AND RESULTS: Five methods were evaluated and fine-tuned for extraction of RNA from Cms. The most efficient non-commercial RNA extraction method included an enzymatic breakdown of the cell wall followed by a phenol extraction. AmpliDet RNA enabled detection of 10,000 molecules of purified rRNA per reaction and 100 cfu of Cms per reaction in more complex samples. Two primer pairs were tested with DNA and RNA purified from Cms. One primer pair was able to distinguish live from dead cells. CONCLUSIONS: An AmpliDet RNA was developed which enabled fast and specific detection of viable cells of Cms in complex substrates at a detection limit of 100 cfu per reaction. SIGNIFICANCE AND IMPACT OF THE STUDY: This novel AmpliDet RNA is carried out in sealed tubes, thus reducing the risk of carry-over contamination. The method will be particularly suitable for studies on the epidemiology of Cms in which viable cells should be exclusively detected.     

Glycoalkaloid aglycone accumulations associated with infection by Clavibacter michiganensis ssp. sepedonicus in potato species Solanum acaule and Solanum tuberosum and their interspecific somatic hybrids

Solanum acaule Bitt., a wild potato species, is closely related to cultivated potato (Solanum. tuberosum L.). Incorporation of desirable traits from allotetraploid [2n=4x=48, 2 endosperm balance number (EBN)] S. acaule (acl) into autotetraploid (2n=4x=48, 4EBN) S. tuberosum (tbr) is difficult due to incongruity boundaries. In this study, three hybrid combinations, each with a specific genome constitution, were produced through protoplast fusion: (1) hexaploid 2x acl (+) 4x tbr, (2) tetraploid 2x acl (+) 2x tbr, and (3) hexaploid 4x acl (+) 2x tbr hybrids. In terms of glycoalkaloid aglycones, the hybrids produced demissidine, tomatidine and solanidine, similarly to the S. acaule parental species, but S. tuberosum synthesised only solanidine. Inoculations with Clavibacter michiganensis ssp. sepedonicus (Cms), which is the causal agent of bacterial ring rot in potato, yielded significantly lower total glycoalkaloid aglycone accumulation both in S. acaule plants and in interspecific hybrids in comparison with the corresponding mock-inoculated plants. However, in S. tuberosum the aglycone levels were either higher or unchanged as a result of infection by Cms. To incorporate the desirable traits of the interspecific somatic hybrids into 4EBN S. tuberosum, sexual backcrosses were carried out. The hexaploid 4x acl (+) 2x tbr hybrids with the hypothetical 4EBN showed the greatest capacity to undergo backcrosses with S. tuberosum.     

A quantitative and direct PCR assay for the subspecies-specific detection of Clavibacter michiganensis subsp. michiganensis based on a ferredoxin reductase gene

The Gram-positive bacterium Clavibacter michiganensis subsp. michiganensis is the causal agent of canker disease in tomato. Because it is very important to control newly introduced inoculum sources from commercial materials, the specific detection of this pathogen in seeds and seedlings is essential for effective disease control. In this study, a novel and efficient assay for the detection and quantitation of C. michiganensis subsp. michiganensis in symptomless tomato and red pepper seeds was developed. A pair of polymerase chain reaction (PCR) primers (Cmm141F/R) was designed to amplify a specific 141 bp fragment on the basis of a ferredoxin reductase gene of C. michiganensis subsp. michiganensis NCPPB 382. The specificity of the primer set was evaluated using purified DNA from 16 isolates of five C. michiganensis subspecies, one other Clavibacter species, and 17 other reference bacteria. The primer set amplified a single band of expected size from the genomic DNA obtained from the C. michiganensis subsp. michiganensis strains but not from the other C. michiganensis subspecies or from other Clavibacter species. The detection limit was a single cloned copy of the ferredoxin reductase gene of C. michiganensis subsp. michiganensis. In conclusion, this quantitative direct PCR assay can be applied as a practical diagnostic method for epidemiological research and the sanitary management of seeds and seedlings with a low level or latent infection of C. michiganensis subsp. michiganensis.     

A Simple and Fast Extraction Procedure to Obtain Amplifyable DNA from Ralstonia (Pseudomonas) Solanacearum and Clavibacter michiganensis ssp. sepedonicus Inoculated Potato Tuber Extracts and Naturally Infected Tubers to Conduct a Polymerase Chain Reaction (PCR)

A modified NaOH alkaline boiling procedure using a mixture of lysozyme and proteases combined with minimized TRIS/HCl/BSA buffer volume was applied to extract amplifyable DNA from the two quarantine bacteria Ralstonia (Pseudomonas) solanacearum and Clavibacter michiganensis ssp. sepedonicus artificially added to potato tuber extracts of a low and a high starch potato variety. A PCR detection threshold of 10⁴−10⁵ colony forming units per ml extract of each quarantine bacterium was obtained by using the two potato varieties, the high starch potato variety resulting in a lower detection threshold.
Similar sensitivities could be obtained from potato tubers naturally infected with both quarantine bacteria. When comparing a published DNA extraction procedure suitable for Clavibacter michiganensis ssp. sepedonicus with the alkaline extraction the latter is much faster and simpler with similar detection thresholds and representing an inexpensive method to obtain suitable template DNA for routine PCR tests.     

Properties of extracellular polysaccharides of potato ring rot pathogen and corresponding recognition sites of potato cell walls

The properties of extracellular polysaccharides of the potato ring rot pathogen Clavibacter michiganensis subsp. sepedonicus (Cms) and the corresponding recognition sites isolated from cell walls of potato suspension cultures have been studied. Extracellular polysaccharides of Cms consist of 4-6 components, which differ greatly in molecular mass (from <1 kD to >700 kD), and are capable of formation of associates stabilized by electrostatic interactions in the presence of calcium. Using affinity column chromatography, sites possessing affinity for the total extracellular polysaccharide complex of Cms were isolated from cell walls of suspension cultures of three potato varieties with different resistance to the pathogen. The content of the receptor sites consisting of glycopeptides and sugars for the variety devoid of resistance was 10 times greater than that for the resistant variety. In the receptor fraction for the latter variety, only sugars were found. The molecular masses of the components of the receptor fraction of cell walls were from 39 to 86 kD. Polypeptides in the recognition sites for the resistant variety escaped detection in electrophoretic patterns. Study of the amino acid composition of the receptor sites of cell walls showed that the sites of the resistant variety contained trace amounts of only seven amino acids. In the sites of the receptive variety 14 amino acids were found, the content of polar amino acids being twice as large as that of nonpolar amino acids. Among polar amino acids, glutamine and glycine prevailed, whereas among nonpolar amino acids valine was dominant. We suggest that one of the reasons of variety-specific resistance of potato to Cms is the absence or a low content of the sites revealing the affinity for bacterial extracellular polysaccharides on the plant cell surface.     

马铃薯内生细菌的分离及其对环腐病菌的拮抗作用检测(简报)

马铃薯是世界四大作物之一,我国是世界上马铃薯种植面积最大的国家,种植面积达6000万亩。近年来,在马铃薯经济良好发展的带动下,马铃薯生产在种植面积和单产水平上有了大幅度提高。然而各种病害随之表现出逐年上升的趋势。环腐病、黑胫病、干腐病是造成北方马铃薯田间和窖存烂薯的主要病害,一般经济损失达20%—30%,严重年份可达50%。马铃薯环腐病害难以防治是因为病菌侵染部     

马铃薯内生细菌的分离及其对环腐病菌的拮抗作用检测（简报）

马铃薯是世界四大作物之一,我国是世界上马铃薯种植面积最大的国家,种植面积达6000万亩。近年来,在马铃薯经济良好发展的带动下,马铃薯生产在种植面积和单产水平上有了大幅度提高。然而各种病害随之表现出逐年上升的趋势。     

A highly efficient transposon mutagenesis system for the tomato pathogen Clavibacter michiganensis subsp. michiganensis.

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 x 10(6) transformants per microg of DNA were generally obtained. Electroporation of C. michiganensis subsp. michiganensis with a suicide vector carrying transposon Tn1409C resulted in approximately 1 x 10(3) transposon mutants per pg 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.     

Effect of composting and pasteurisation on two important quarantine pests of potato

The effect of composting and pasteurization on the quarantine pests of potato Clavibacter michiganensis ssp. sepedonicus (Cms) and Synchytrium endobioticum (Se) were examined on an experimental scale. Composting was performed with 2-L pots and 60-L composters for two months at temperatures below 50 degrees C and for 12 and 21 days at temperatures above 65 degrees C. Pasteurization was performed via water bath at 70 degrees C for maximum 2 hours. Pathogens were introduced directly or via carriers into the processes. After composting for two months and for 12 and 21 days it was possible to isolate vital Cms cells from bioassay plants and vital resting spores of Se could be extracted from sample material. Likewise it was possible to isolate vital Cms cells and resting spores of Se after pasteurization for up to two hours. Both pests could not be killed completely during the performed processes. Further studies concerning sanitization of potato wastes are necessary.