Bacterial Ring Rot Of Potato Research Articles

Influence of the high infectious load of the causal agent of bacterial ring rot of potatoes on variations in hydrogen peroxide and cAMP levels in transgenic potato plants <i>in vitro</i> with increased contents of endogenous hydrogen peroxide

Effects of the infectious load of the causal agent Clavibacter michiganensis sps. Sepedonicus (Cms) of ring rot of potatoes on hydrogen peroxide and cAMP levels in potato plants in vitro were studied on three transgenic lines of the Scarb cultivar: line L17.2 – plants transformed with the native gene of glucose oxidase gox from the highly active fungal strain Penicillium funiculosum 46.1; line M7.3 – plants transformed with the modified gene gox-mod; and line Pb14.10 – plants transformed with a vector without a target gene. In addition, non-transgenic plants of the same cultivar (medium resistant against Cms), Lugovskaya (resistant against Cms) and Lukyanovsky (susceptible to Cms) were examined. In the plants infected with Cms (0.2х10 8 cells/ml), the dynamics of cAMP and Н2О2 indicated a balanced system signal “root–stem”. The Cms titre (2х10 8 cells/ml) caused a systemic increase in the concentration of Н 2 О 2 in the organs of almost all plant cultivars; however, a systemic increase in the level of cAMP occurred only in the Lugovskaya cultivar. In all the studied transgenic lines, cAMP levels remained at the control level, accompanied by both local and extensive necrosis. In transgenic potato lines, the observed decrease in protective responses (both local and extensive necrosis) is likely to be associated with an extremely high baseline level of endogenous hydrogen peroxide and an imbalance in cAMP levels. The introduction of the gox gene into potato plants was found to increase their resistance against high Cms titres.

Bacterial ring rot of potato caused by Clavibacter sepedonicus: Asuccessful example of defeating the enemy under international regulations.

BackgroundBacterial ring rot of potato (Solanum tuberosum) caused by the gram‐positive coryneform bacterium Clavibacter sepedonicus is an important quarantine disease threatening the potato industry around the globe. Since its original description in 1906 in Germany, management of ring rot has been a major problem due to the seedborne nature (via seed tubers not true seeds) of the pathogen allowing the bacterium to be transmitted long distances via infected tubers.Disease symptomsOn growing potato plants: interveinal chlorosis on leaflets leading to necrotic areas and systemic wilt. On infected tubers: vascular tissues become yellowish brown with a cheesy texture due to bacterial colonization and decay.Host rangePotato is the main host of the pathogen, but natural infection also occurs on eggplant, tomato, and sugar beet.Taxonomic status of the pathogenClass: Actinobacteria; Order: Actinomycetales; Family: Microbacteriaceae; Genus: Clavibacter; Species: Clavibacter sepedonicus (Spieckermann and Kotthoff 1914) Li et al. 2018.Synonyms (nonpreferred scientific names) Aplanobacter sepedonicus; Bacterium sepedonicum; Corynebacterium sepedonicum; Corynebacterium michiganense pv. sepedonicum; Clavibacter michiganensis subsp. sepedonicus.Microbiological propertiesGram‐positive, club‐shaped cells with creamy to yellowish‐cream colonies for which the optimal growth temperature is 20–23°C.DistributionAsia (China, Japan, Kazakhstan, Nepal, North Korea, Pakistan, South Korea, Uzbekistan, the Asian part of Russia), Europe (Belarus, Bulgaria, Czech Republic, Estonia, Finland, Georgia, Germany, Greece, Hungary, Latvia, Lithuania, Norway, Poland, Romania, European part of Russia, Slovakia, Spain, Sweden, Turkey, Ukraine), and North America (Canada, Mexico, USA).Phytosanitary categorizationCORBSE: EPPO A2 list no. 51. EU; Annex designation I/A2.

Pest categorisation of Clavibactersepedonicus.

Following a request from the European Commission, the EFSA Panel on Plant Health performed a pest categorisation of Clavibacter sepedonicus, a well‐defined and distinguishable bacterial plant pathogen of the family Microbacteriaceae. C. sepedonicus causes bacterial ring rot of potato and is reported from North America, Asia and Europe. The bacterium is mostly tuber transmitted, but it can also enter host plants through wounds or via contaminated equipment. C. sepedonicus is regulated in Council Directive 2000/29/EC (Annex IAII, as Clavibacter michiganensis subsp. sepedonicus) as a harmful organism whose introduction into the EU is banned. In addition, Council Directive 1993/85/EEC concerns the measures to be taken within EU Member States (MS) against C. sepedonicus to (a) detect it and determine its distribution, (b) prevent its occurrence and spread, and (c) control it with the aim of eradication. The pest is present in several EU MS, but in all cases with a restricted distribution and under official control. C. sepedonicus could enter the EU and spread primarily via host plants for planting (i.e. potato tubers). The pest could establish in the EU, as the main host (potato) is commonly grown and climatic conditions are favourable. Direct potato losses following infection by C. sepedonicus can be substantial and are due to the destruction of the vascular tissue, wilting of the plant and rotting of the tubers. Infected hosts can remain asymptomatic. The main knowledge gaps are the geographic distribution of the pest and the host range. The criteria assessed by the Panel for consideration of C. sepedonicus as a potential quarantine pest are met, while, for regulated non‐quarantine pests, the criterion on the widespread presence in the EU is not met.

Analysis of Extreme Phenotype Bulk Copy Number Variation (XP-CNV) Identified the Association of rp1 with Resistance to Goss's Wilt of Maize.

Goss's wilt (GW) of maize is caused by the Gram-positive bacterium Clavibacter michiganensis subsp. nebraskensis (Cmn) and has spread in recent years throughout the Great Plains, posing a threat to production. The genetic basis of plant resistance is unknown. Here, a simple method for quantifying disease symptoms was developed and used to select cohorts of highly resistant and highly susceptible lines known as extreme phenotypes (XP). Copy number variation (CNV) analyses using whole genome sequences of bulked XP revealed 141 genes containing CNV between the two XP groups. The CNV genes include the previously identified common rust resistant locus rp1. Multiple Rp1 accessions with distinct rp1 haplotypes in an otherwise susceptible accession exhibited hypersensitive responses upon inoculation. GW provides an excellent system for the genetic dissection of diseases caused by closely related subspecies of C. michiganesis. Further work will facilitate breeding strategies to control GW and provide needed insight into the resistance mechanism of important related diseases such as bacterial canker of tomato and bacterial ring rot of potato.

Volatile organic compounds produced by a soil-isolate, Bacillus subtilis FA26 induce adverse ultra-structural changes to the cells of Clavibacter michiganensis ssp. sepedonicus, the causal agent of bacterial ring rot of potato.

Rhizobacterial volatile organic compounds (VOCs) play an important role in the suppression of soil-borne phytopathogens. In this study, the VOCs produced by a soil-isolate, Bacillus subtilis FA26, were evaluated in vitro for their antibacterial activity against Clavibacter michiganensis ssp. sepedonicus (Cms), the causal agent of bacterial ring rot of potato. The VOCs emitted by FA26 inhibited the growth of Cms significantly compared with the control. Scanning and transmission electron microscopy analyses revealed distorted colony morphology and a wide range of abnormalities in Cms cells exposed to the VOCs of FA26. Varying the inoculation strategy and inoculum size showed that the production and activity of the antibacterial VOCs of FA26 were dependent on the culture conditions. Headspace solid-phase microextraction/gas chromatography-mass spectrometry analyses revealed that FA26 produced 11 VOCs. Four VOCs (benzaldehyde, nonanal, benzothiazole and acetophenone) were associated with the antibacterial activity against Cms. The results suggested that the VOCs produced by FA26 could control the causal agent of bacterial ring rot of potato. This information will increase our understanding of the microbial interactions mediated by VOCs in nature and aid the development of safer strategies for controlling plant disease.

Inhibitory activity of Athyrium sinense extracts against Clavibater michiganense subsp. sepedonicum

Clavibater michiganense subsp. sepedonicum , the causal agent of a serious disease called bacterial ring rot of potato, is widespread under both greenhouse and field cultivation. In order to find a promising method for the control of this disease, Athyrium sinense extracts were investigated in the study presented here. Results showed that the A. sinense extracts had significantly inhibitory activity against C. michiganense subsp. sepedonicum . Eight organic solvents were used to obtain A. sinense extracts, and ethanol was selected as an ideal extraction solvent. Based on single factor experiment results and L 27 3 (13) orthogonal experiment results, optimum extraction conditions were obtained. They were A 2 B 3 C 3 , solid to liquid ratio 1:20 (g:mL), temperature 97°C and extraction time 10h. Stability assays revealed that Zn 2+ , Fe 3+ , acidic and neutral conditions enhanced inhibitory activity, and extracts were stable under ultraviolet (UV). Transmission electron microscopy (TEM) showed drastic changes caused by A. sinense extracts, including lack of cytoplasmic materials, formation of vacuoles and bacterial deformation. These results revealed A. sinense extracts have strong inhibitory activity against C. michiganense subsp. sepedonicum , and can be used as a potent phytochemical fungicide.

Efficacy of agricultural disinfectants on biofilms of the bacterial ring rot pathogen, Clavibacter michiganensis subsp. sepedonicus

Susceptibility of biofilms of Clavibacter michiganensis subsp. sepedonicus, the causal agent of bacterial ring rot of potato, to three common agricultural disinfectants was tested. The MBECTM assay device was used to evaluate optimum parameters for growing artificial biofilms. These conditions were determined to be 7 days of growth at 23°C in a yeast extract-glucose-mineral salts medium. As expected, the bacteria in the biofilm state were more resistant to disinfection by chemical treatment with sodium hypochlorite, quaternary ammonium, and hydrogen peroxide when compared with planktonic cells. Artificial biofilms were also grown on five different surface materials typically found in commercial potato storage facilities (concrete, mild steel, rubber, polycarbonate and wood) using the BESTTM assay device to test the effect of surface type on biofilm susceptibility to disinfection. Sodium hypochlorite was the most effective disinfectant on the wood surface and hydrogen peroxide was best on the mild steel surface. Efficacies of the various disinfectants were not significantly different on concrete, rubber and polycarbonate surfaces. When artificially grown biofilms and those grown naturally in potato tissue were transferred to, and dried onto, coupons of the different surface materials, they were significantly more difficult to inactivate than in situ grown biofilms. The resistance of plant pathogenic bacteria in the biofilm state, particularly when spread and dried onto surfaces of agricultural machines and other equipment, to commonly used disinfectants, has important implications for disease control strategies that depend largely on strict sanitary and hygienic practices.

Antibacterial activity of petroleum ether fraction from Laminaria japonica extracts against Clavibacter michiganensis subsp. sepedonicus

We prepared an ethanol extract from Laminaria japonica, and obtained five fractions from the extract using various solvents. We investigated the antibacterial activities of the fractions against Clavibacter michiganensis subsp. sepedonicus (Spieckermann & Kotthoff) Davis et al., the causal agent of bacterial ring rot of potato (BRR). Among all the fractions, the petroleum ether fraction showed the strongest (P < 0.05) antibacterial activity against C. michiganensis subsp. sepedonicus. We analyzed the petroleum ether fraction to detect the components with antibacterial activity, and identified 24 compounds. The main constituents of the petroleum ether extract were esters (35.28 %) and acids (44.84 %). Cell membrane permeability assays, scanning electron microscopy (SEM) and transmission electron microscopy (TEM) analyses revealed that the petroleum ether fraction caused drastic ultrastructural changes in the cells of the pathogen. The petroleum ether fraction caused envelope disruption, cell deformation, the separation of the cell wall from the cell membrane, and the formation of vacuoles. These results clearly indicate that the petroleum ether fraction obtained from L. japonica shows strong antibacterial activity against the causal agent of BRR.

Laminaria japonica Extract, an Inhibitor of Clavibater michiganense Subsp. Sepedonicum.

Bacterial ring rot of potato is one of the most serious potato plant and tuber diseases. Laminaria japonica extract was investigated for its antimicrobial activity against Clavibater michiganense subsp. sepedonicum (Spieckermann & Kotthoff) Davis et al., the causative agent of bacterial ring rot of potato. The results showed that the optimum extraction conditions of antimicrobial substances from L. japonica were an extraction temperature of 80°C, an extraction time of 12 h, and a solid to liquid ratio of 1∶25. Active compounds of L. japonica were isolated by solvent partition, thin layer chromatography (TLC) and column chromatography. All nineteen fractionations had antimicrobial activities against C. michiganense subsp. sepedonicum, while Fractionation three (Fr.3) had the highest (P<0.05) antimicrobial activity. Chemical composition analysis identified a total of 26 components in Fr.3. The main constituents of Fr.3 were alkanes (80.97%), esters (5.24%), acids (4.87%) and alcohols (2.21%). Antimicrobial activity of Fr.3 against C. michiganense subsp. sepedonicum could be attributed to its ability to damage the cell wall and cell membrane, induce the production of reactive oxygen species (ROS), increase cytosolic Ca2+ concentration, inhibit the glycolytic pathway (EMP) and tricarboxylic acid (TCA) cycle, inhibit protein and nucleic acid synthesis, and disrupt the normal cycle of DNA replication. These findings indicate that L. japonica extracts have potential for inhibiting C. michiganense subsp. sepedonicum.

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

It has previously been shown that the tomato pathogen Clavibacter michiganensis subsp. michiganensis secretes a 14-kDa protein, C. michiganensis subsp. michiganensis AMP-I (CmmAMP-I), that inhibits growth of Clavibacter michiganensis subsp. sepedonicus, the causal agent of bacterial ring rot of potato. Using sequences obtained from tryptic fragments, we have identified the gene encoding CmmAMP-I and we have recombinantly produced the protein with an N-terminal intein tag. The gene sequence showed that CmmAMP-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.

Protective effect of Polygonum orientale L. extracts against Clavibater michiganense subsp. sepedonicum, the causal agent of bacterial ring rot of potato.

The Polygonum orientale L. extracts were investigated for antibacterial activity against Clavibater michiganense subsp. sepedonicum (Spieckermann & Kotthoff) Davis et al., the causal agent of a serious disease called bacterial ring rot of potato. The results showed that the leaf extracts of P. orientale had significantly (p<0.05) greater antibacterial activity against C. michiganense subsp. sepedonicum than root, stem, flower extracts in vitro. According to the results of single factor experiments and L273(13) orthogonal experiments, optimum extraction conditions were A1B3C1, extraction time 6 h, temperature 80°C, solid to liquid ratio 1∶10 (g:mL). The highest (p<0.05) antibacterial activity was observed when pH was 5, excluding the effect of control. The extracts were stable under ultraviolet (UV). In vivo analysis revealed that 50 mg/mL of P. orientale leaf extracts was effective in controlling decay. Under field conditions, 50 mg/mL of P. orientale leaf extracts also improved growth parameters (whole plant length, shoot length, root length, plant fresh weight, shoot fresh weight, root fresh weight, dry weight, and number of leaves), in the 2010 and 2011 two growing seasons. Further solvent partition assays showed that the most active compounds were in the petroleum ether fractionation. Transmission electron microscopy (TEM) showed drastic ultrastructural changes caused by petroleum ether fractionation, including bacterial deformation, electron-dense particles, formation of vacuoles and lack of cytoplasmic materials. These results indicated that P. orientale extracts have strong antibacterial activity against C. michiganense subsp. sepedonicum and a promising effect in control of bacterial ring rot of potato disease.

The Putative Secreted Serine Protease Chp-7 Is Required for Full Virulence and Induction of a Nonhost Hypersensitive Response by Clavibacter michiganensis subsp. sepedonicus

Molecular biological studies on Clavibacter michiganensis subsp. sepedonicus, the causal agent of bacterial ring rot of potato, have gained greater feasibility due to the recent availability of whole genomic sequences and genetic tools for related taxa. Here, we describe the first report of construction and characterization of a transposon (Tn) mutant library of C. michiganensis subsp. sepedonicus sp. strain R10. Since virulence of R10 in potato has been shown previously to be associated with elicitation of a nonhost hypersensitive response (HR), the mutant library was screened initially for loss of HR in tobacco. The screen identified two HR-negative mutants containing Tn insertions within the same gene, CMS2989 (chp-7), although at distinct locations. chp-7 is one of 11 pat-1 homologs in C. michiganensis subsp. sepedonicus. HR-negative mutants of R10 multiplied to the same extent as wild type in planta but were less virulent in potato. Complementation with chp-7 restored virulence as well as the HR phenotype. Together, these findings demonstrate a role for chp-7 in C. michiganensis subsp. sepedonicus-plant interactions.

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

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.

Epidemiology of bacterial ring rot of potato in plant and soil–consequences for disease management

In five field experiments, carried out from 2002 to 2004, the infestation of potato plants and the contamination of soil with Clavibacter michiganensis subsp. sepedonicus, the causal agent of bacterial ring rot of potato, were investigated throughout the growing season. Aiming at a detailed description of disease progress, for the first time potato tubers, stems and roots as well as soil were examined in parallel. The development of bacterial populations in plant organs and soil differed considerably within and between the experiments. Roots showed the highest and tubers the lowest incidence of infection of plant organs based on polymerase chain reaction (PCR) and immunofluorescence antibody staining (IFAS). Logistic regression analyses indicated a significant increase in the probability of infestation (in the case of soil: contamination) during the growing season for all investigated plant parts of highly infected plants and the respective soil samples. Results were discussed in terms of the suitability of particular plant organs and the determination of the best sampling time point for official testing. Furthermore, the risks are evaluated to contaminate machinery on the field and to spread bacterial ring rot through soil.

History of bacterial ring rot of potato in the Czech Lands and a proposal for relaxation of strict quarantine measures

In the supposed or proven incidence of bacterial ring rot caused by &lt;i&gt;Clavibacter michiganensis&lt;/i&gt; subsp. &lt;i&gt;sepedonicus&lt;/i&gt; (&lt;i&gt;Cms&lt;/i&gt;) in certified seed and commercial potatoes, five periods can be identified in the Czech Lands from 1910 to 2006: (&lt;i&gt;i&lt;/i&gt;) high incidence of &lt;i&gt;Cms&lt;/i&gt; in potato crops is claimed (about 1910–1929); (&lt;i&gt;ii&lt;/i&gt;) very low incidence in certified potatoes and sporadic occurrence in commercial potatoes (about 1930–1985); (&lt;i&gt;iii&lt;/i&gt;) increasing incidence of &lt;i&gt;Cms&lt;/i&gt; in certified seed potatoes and its sporadic occurrence in commercial potatoes is assumed (about 1986–1997); (&lt;i&gt;iv&lt;/i&gt;) a relatively high percentage of potato tuber samples proved to be infected by &lt;i&gt;Cms&lt;/i&gt;, namely 1.14% in seed potatoes in 1998 and 4.13% in commercial potatoes in 1999 (1998–2004 period); (&lt;i&gt;v&lt;/i&gt;) progressive decrease of &lt;i&gt;Cms&lt;/i&gt; incidence to zero in seed potato samples and 0.19% in commercial samples in 2005, followed by a slight increase to 0.15% in seed potatoes and 0.23% in commercial potatoes in 2006 (2005–2006 period). Thus, up to 2006, &lt;i&gt;Cms&lt;/i&gt; was and is not widely distributed in the CR and is actively and effectively controlled, mainly through the zero tolerance for ring rot bacterium in the seed potato certification program. In the CR, &lt;i&gt;Cms&lt;/i&gt; has a relatively low capacity for damage and can hardly be considered as a pest of national economic importance. Strictly speaking, &lt;i&gt;Cms&lt;/i&gt; does not fulfill the criteria for a quarantine organism. If, however, the quarantine status of &lt;i&gt;Cms&lt;/i&gt; will be maintained, the severe post-entry measures against it should be relaxed.

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

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. 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. Clavibacter michiganensis ssp. michiganensis produces various antibacterial substances that are active against Cms. 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.

Fate of Clavibacter michiganensis ssp. sepedonicus, the Causal Organism of Bacterial Ring Rot of Potato, in Weeds and Field Crops

Crops and weeds were tested for their ability to host Clavibacter michiganensis ssp. sepedonicus (Cms), the causal agent of bacterial ring rot in potato. Ten crops grown in rotation with potato in Europe, namely maize, wheat, barley, oat, bush bean, broad bean, rape, pea and onion and five cultivars of sugar beet were tested by stem and root inoculation. About 6–8 weeks after inoculation, Cms could be detected in most crops except onion and sugar beet, in larger numbers in stems (105–106 cells/g of tissue) than in roots (≤103 cells/g of tissue) in immunofluorescence cell-staining (IF). Cms was successfully re-isolated only from IF-positive stem samples of maize, bush bean, broad bean, rape and pea, but not from roots. Twelve solanaceous weeds and 13 other weeds, most commonly found in potato fields in Europe, were tested in IF as hosts of Cms by stem and root inoculations. Only in Solanum rostratum, a weed present in northern America, Cms persisted in high numbers (108 cells/g tissue) in stems and leaves, where it caused symptoms. In the other solanaceous weeds, Cms persisted at low numbers (approximately 105 cells/g of tissue) in stems but less so in roots. The bacteria could be frequently re-isolated from stem but not from root tissues. In 2 consecutive years, plants from 14 different weed species were collected from Cms-contaminated potato field plots and tested for the presence of Cms by dilution plating or immunofluorescence colony-staining (IFC), and by AmpliDet RNA, a nucleic acid-based amplification method. Cms was detected in roots but not in stems of Elymus repens plants growing through rotten potato tubers, and in some Viola arvensis and Stellaria media plants, where they were detected both in stems and roots, but more frequently by AmpliDet RNA than by IFC.

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

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 (&amp;gt; 0.5 mg/ml), weakly with proteins from cell walls (&amp;gt; 200 &amp;mu;g/ml) and with whole homologous antigen (concentration 104&amp;ndash;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.

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

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.

In Planta - Complementation of Clavibacter Michiganensis Subsp. sepedonicus Strains Deficient in Cellulase Production or HR Induction Restores Virulence

Clavibacter michiganensis subsp. sepedonicus, a Gram positive bacterium that causes bacterial ring rot of potato, was studied in eggplant, an alternate host, using strains that differed in phenotype. Two factors affecting virulence, the ability to induce a hypersensitive response (HR) and cellulase production, were studied. A plasmid-free isolate of C. michiganensis subsp. sepedonicus that causes HR on tobacco but is unable to produce cellulase multiplied efficiently in planta, but caused only weak symptoms. In contrast, a strain that is unable to induce HR on tobacco but produces cellulase was impaired in the ability to multiply in the host and caused no symptoms. When the two non-virulent strains were coinoculated into eggplants, typical disease symptoms developed. This enhancement was not due to formation of a new phenotype or significant increases in population density of either of the strains. Our results suggest that both cellulase production and the ability to induce HR are required for a successful infection process and disease induction by C. michiganensis subsp. sepedonicus. Our results additionally suggest that the ability to induce HR on non-host plants is required for multiplication in the host plant, whereas cellulase expression is necessary for induction of disease symptoms.