Evaluation of the API 50CH and API ZYM systems for rapid characterization of Clavibacter michiganensis subsp. sepedonicus, causal agent of potato ring rot

API 50CH and API ZYM systems were used to characterize fifty-three strains of Clavibacter michiganensis subsp. sepedonicus from different geographic locations and several reference strains of the same and different species, including other potato pathogens. Clavibacter michiganensis subsp. sepedonicus strains displayed a high level of homogeneity, both in carbohydrate utilization and in enzymatic activity. Using API 50CH and API ZYM it was possible to differentiate C. michiganensis subsp. sepedonicus strains from the remaining taxa analysed in this study, which included representative strains of the other subspecies of C. michiganensis as well as other bacterial pathogens affecting potatoes. Therefore, these systems could be used as an effective method to characterize C. michiganensis subsp. sepedonicus. Such a procedure would constitute an alternative system to the conventional nutritional and physiological identification tests currently included in the official methods employed in the European Union to detect and identify this bacterium. The results obtained with the API systems agreed with the current taxonomic classification of C. michiganensis, clearly separating sepedonicus from the other subspecies belonging to this species.     

Repetitive Sequence-derived PCR Profiling Using the BOX-A1R Primer for Rapid Identification of the Plant Pathogen Clavibacter Michiganensis Subspecies Sepedonicus

Repetitive sequence-derived PCR using the BOX-A1R primer was used to generate genomic fingerprints of Clavibacter michiganensis subspecies sepedonicus, the causal agent of bacterial ring rot disease of potato. A total of 35 C. michiganensis subsp. sepedonicus strains were selected for study in order to represent the widest possible historical, morphological and geographical diversity of the organism. Comparison was made with genomic fingerprints of C. michiganensis subsp. michiganensis, C. michiganensis subsp. insidiosus, C. michiganensis subsp. tessellarius, C. michiganensis subsp. nebraskensis as well as other related Gram positive plant pathogens. The resultant genomic fingerprints and subsequent cluster analysis show C. michiganensis subsp. sepedonicus to form a remarkably homogeneous group with approximately 84% similarity between all of the strains tested. There was no evidence to suggest that fingerprints varied with historic, morphological or geographic diversity. In addition, C. michiganensis subsp. sepedonicus isolated from asymptomatic sugar beet had the same fingerprint as those which were isolated as potato pathogens. This group was easily distinguished from the clusters formed by the other subspecies of C. michiganensis and Gram positive plant pathogens. The potential for this technique to be used as a relatively rapid method to replace the time consuming and sometimes inconclusive eggplant bioassay test is discussed.     

Rapid Identification of Clavibacter michiganensis Subspecies Sepedonicus based on the Stable Low Molecular Weight RNA (LMW RNA) Profiles

Clavibacter michiganensis subsp. sepedonicus causes potato ring rot disease. The identification process for this bacterium is complex and long. This work demonstrates that the stable low-molecular-weight (LMW) RNA profiles allow their rapid identification. Staircase electrophoresis in polyacrylamide gels was used to analyze the LMW RNA profiles of 54 strains of C. michiganensis subsp. sepedonicus from different geographic origins. The profiles of several strains of other subspecies of C. michiganensis and other pathogens of potatoes were also analyzed. All the strains of C. michiganensis subsp. sepedonicus had the same LMW RNA profile. They had a band in class 2 of tRNA that was absent in the other subspecies of the species C. michiganensis. Also, the LMW RNA of C. michiganensis subsp. sepedonicus was different with respect to the LMW RNA profiles of other pathogens of potato. The results indicate the possible utilization of LMW RNA profiles in identification of the bacteria causing potato ring rot disease.     

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.     

Evaluation of antagonistic bacteria for suppression of bacterial ring rot of potato

Bacterial strains with potential for biological control of bacterial ring rot of potato caused byClavibacter michiganensis subsp.sepedonicus were isolated from the surface of potato tubers. Eighty-eight potential biocontrol candidates, selected on the basis ofin vitro antibiosis toC. m. sepedonicus, produced inhibition zones with radii ranging from 0.5 to 16 mm on test plates. All antagonistic isolates were screened in the greenhouse for biocontrol activity on micropropagated potato plantlets root-inoculated withC. m. sepedonicus. Eight strains consistently prevented infection of plantlets but there was no significant correlation between the width of the inhibition zone in thein vitro assay and ring rot suppression in the plant bioassay. Three strains that showed a high level of biological control potential were identified as a saprophytic enteric bacterium (strain 7G), anArthrobacter sp. (strain 16C), and a soil coryneform bacterium (strain 18A). These were tested in a field plot by co-inoculating cut seed potato tubers withC. m. sepedonicus and antagonists. Strains 7G and 18A significantly increased plant stand whereas 16C decreased disease incidence. The relative number of ostensibly ring rot-free progeny tubers was generally greater when antagonists were present.     

植物病原棒形杆菌属分类最新进展

植物病原棒形杆菌属Clavibacter的分类随着研究的深入和鉴定技术水平的不断进步一直在发生着变化,之前普遍认可的观点为该属仅含有1个种,即密执安棒形杆菌C. michiganensis,种下又分为5个亚种,分别为密执安亚种C. michiganensis subsp. michiganensis、诡橘亚种C. michiganensis subsp. insidiosus、尼布拉斯加亚种C. michiganensis subsp. nebraskensis、环腐亚种C. michiganensis subsp. sepedonicus和花叶亚种C. michiganensis subsp. tessellarius。最近几年又有4个新亚种陆续被报道,分别为菜豆亚种C. michiganensis subsp. phaseoli、辣椒亚种C. michiganensis subsp. capsici、加利福尼亚亚种C. michiganensis subsp. californiensis和智利亚种C. michiganensis subsp. chilensis。随着全基因组分析和多基因分析技术在细菌分类上的应用,之前被广泛认可的4个亚种(诡橘亚种、尼布拉斯加亚种、环腐亚种和花叶亚种)及新亚种辣椒亚种也曾被建议定义为种,即C. insidiosus、C. nebraskensis、C. sepedonicus、C. tessellarius和C. capsici。为便于研究人员了解该属最新的分类研究现状,本文对植物病原棒形杆菌属的分类历史及最新分类现状进行了系统梳理,期望为该属病原菌的风险评估、检测鉴定等研究提供分类学参考。     

Effect of phytotoxic compounds produced by Clavibacter michiganensis subsp. michiganensis on resistant and susceptible tomato plants

A phytotoxic fraction of high molecular weight was isolated from the culture filtrate ofClavibacter michiganensis subsp.michiganensis, the causal agent of bacterial canker of tomato, and partly purified. This high molecular weight fraction consists of sugars and a minor protein moiety and is therefore probably of similar nature to that of the toxin fromC. michiganensis subsp.michiganensis reported earlier in literature.The high molecular weight fraction was albe to induce wilting, the predominant symptom of the disease, as shown in a bioassay with tomato cuttings. However, this wilting reaction turned out to be non-specific in the bioassay, since (partially) resistant and susceptible genotypes responded similarly. No correlation could be found between the degree of virulence of fiveC. michiganensis subsp.michiganensis strains and the amount of the phytotoxic high molecular weight fraction produced in vitro.As the isolated high molecular weight fraction showed a phytotoxic effect on tomato plants it is worthwhile to test its potential for use as a selective agent in in vitro selection.Samenvatting Een fytotoxische fractie werd geïsoleerd uit cultuurfiltraat vanClavibacter michiganensis subsp.michiganensis, de veroorzaker van de bacterieverwelkingsziekte bij tomaat. Een eerste karakterisering toonde aan dat deze toxische fractie hoog-moleculaire component(en) bevat, bestaande uit polysacchariden en een gering percentage eiwit. Dit is in overeenstemming met toxines vanC. michiganensis subsp.michiganensis die al eerder beschreven zijn.Deze hoogmoleculaire toxische fractie was in staat verwelking te induceren van stengeltoppen van verschillendeLycopersicon esculentum enL. peruvianum genotypen in een bioassay. Gewichtsverandering van de stengeltoppen, uitgedrukt als percentage ten opzichte van het begingewicht, werd gebruikt als parameter voor verwelking. De toxische fractie reageerde niet-specifiek in de bioassay, want er werd geen verschil gevonden in respons van (partieel) resistente en gevoelige genotypen. Er bleek geen correlatie te zijn tussen de mate van virulentie van verschillende isolaten vanC. michiganensis subsp.michiganensis en de hoeveelheid van de toxische fractie geproduceerd in vitro.Het mogelijke gebruik van deze hoogmoleculaire toxische fractie als selectief agens bij in vitro selectie zal nader onderzocht worden.     

Detection of Clavibacter michiganensis subsp. sepedonicus in Potato Tubers by Multiplex PCR with Coamplification of Host DNA

A new multiplex PCR assay was developed for the detection of Clavibacter michiganensis subsp. sepedonicus in potato tubers. The assay combines two different tests in one reaction mixture. First, a highly specific and sensitive detection of the pathogen and second, an indicator test for successful amplification (internal PCR control), which monitors potentially false-negative PCR results, caused by inhibition of the PCR. For the simultaneous amplification of two different targets in one reaction mixture, a mix of two different primer sets was used. For the detection of C. michiganensis subsp. sepedonicus, a pathogen-specific primer set PSA-1/PSA-R was used, based on the intergenic spacer region of the 16S–23S rRNA genes of C. michiganensis subsp. sepedonicus. For the simultaneous amplification of the internal PCR control, the plant-specific primer set NS-7-F/NS-8-R was employed, permitting amplification of target sequence from plant DNA present in DNA extractions from potato core fluid. The applicability of the multiplex PCR was verified in 3500 composite samples of 200 seed potato tubers from 143 different cultivars in a survey for C. michiganensis subsp. sepedonicus by parallel testing using immunofluorescence, a bioassay in eggplant seedlings and multiplex PCR.     

Analysis of the toxicity of purothionins and hordothionins for plant pathogenic bacteria

Purothionins (PTHs) and hordothionins (HTHs) were purified by cation-exchange chromatography from petroleum-ether extracts of wheat and barley flour respectively. The HTHs could be separated into two fractions, HTH-1 and HTH-2. Radial diffusion assays and micro-plate broth dilution assays with a number of plant pathogenic bacteria showed that these proteins were toxic forClavibacter michiganensis subsp.michiganensis, the causal agent of bacterial canker on tomato,C. m. subsp.sepedonicus, the causal agent of ring rot on potato, andXanthomonas campestris pv.vesicatoria, the causal agent of a spot disease on tomato and pepper. Only minor differences in toxicity between PTHs and HTHs, and between HTH-1 and HTH-2, were detected. Minor differences in toxicity of these thionins were also detected for different strains of these bacteria. The use of these plant proteins for engineering bacterial disease resistance into solanaceous crops will be discussed.     

Polymorphism analysis of housekeeping genes for identification and differentiation of <Emphasis Type="Italic">Clavibacter michiganensis</Emphasis> subspecies

The utility of polymorphism analysis was determined for differentiation of the following subspecies of the Gram-positive plant pathogenic bacterium, Clavibacter michiganensis: C. m. subsp. michiganensis, C. m. subsp. sepedonicus, C. m. subsp. insidiosus C. m. subsp. nebraskensis, and C. m. subsp. tessellarius. Specific primers designed for amplification of the housekeeping genes recA, rpoB, and rpoD generated 827-, 1037-, and 862-bp DNA fragments, respectively. PCR products obtained from 40 C. michiganensis strains were analysed using RFLP with four restriction endonucleases, and those PCR products with specific RFLP patterns were sequenced. The genotypes discriminated after PCR–RFLP were specific for each subspecies and also allowed for differentiation of C. m. subsp. michiganensis strains. Sequence analysis of the recA, rpoB, and rpoD gene fragments also distinguished C. michiganensis subspecies and was useful for phylogenetic analysis of all subspecies. For rapid, inexpensive, and effective differentiation of the five subspecies in this research, we recommend the amplification of recA and/or rpoD gene fragments and digestion of the PCR products with the restriction endonuclease FnuDII.     

Validation of four real‐time TaqMan PCRs for the detection of Ralstonia solanacearum and/or Ralstonia pseudosolanacearum and/or Clavibacter michiganensis subsp. sepedonicus in potato tubers using a statistical regression approach

A new DNA extraction method and a new multiplex real‐time TaqMan PCR test for detection of Ralstonia solanacearum, Ralstonia pseudosolanacearum and Clavibacter michiganensis subsp. sepedonicus in asymptomatic potato tubers are presented. This new multiplex PCR and three published TaqMan PCRs for detection of R. solanacearum and/or R. pseudosolanacearum and/or R. syzygii spp. and/or C. michiganensis subsp. sepedonicus were validated using linear regression analysis for estimating the Ct values and its variation at 5 × 10³ bacteria mL^?1. The three published PCRs that have been validated are Massart et al. (2014, detecting R. solanacearum and C. michiganensis subsp. sepedonicus), Weller et al. (1999, detecting R. solanacearum, R. pseudosolanacearum and R. syzygii spp.) and Gudmestad et al. (2009, detecting C. michiganensis subsp. sepedonicus). All tested PCRs were fit for purpose for their target organisms. The PCR tests have different target genes, allowing one of the sets to be used as first screening test and another as second screening test for the detection of R. solanacearum and/or R. pseudosolanacearum and/or C. michiganensis subsp. sepedonicus in asymptomatic potato tubers.     

Development of Strain-specific Primers for a Strain of Gliocladium catenulatum Used in Biological Control

The randomly amplified polymorphic DNA (RAPD) technique was used to develop strain-specific primers for Gliocladium catenulatum strain J1446, which is promising in biological control. One of the primer pairs developed proved to be strain-specific; strain J1446 was differentiated from 16 G. catenulatum strains and six other strains of two Gliocladium species, as well as from Trichoderma virens, and isolates of Nectria spp. and Fusarium spp. Specific primers were also tested with DNA isolated from cucumber leaves, treated or untreated with a solution made from Gliocladium powder. The expected amplification product was produced only from treated leaves. DNA isolated from Gliocladium-treated potato tubers and fungi grown in peat was also used in amplification reactions. Strain-specific primers detected strain J1446 when the amount of DNA was 5pg or more. Some variation between the Gliocladium strains was found by the random amplified microsatellites method (RAMS) and the universally primed polymerase chain reaction method (UP-PCR), but no clear fragments specific to strain J1446 were produced. Cross-blot hybridisation of UP-PCR products differentiated strain J1446 from T. virens, but not from the Gliocladium isolates. The 28S rDNA sequences and -tubulin sequences were identical or very similar in all Gliocladium strains. Thus, it is possible that the Gliocladium strains of the present study are conspecific, which means that a revision in the taxonomy of Gliocladium species may be necessary.     

Performance of real‐time PCR and immunofluorescence for the detection of Clavibacter michiganensis subsp. sepedonicus and Ralstonia solanacearum in potato tubers in routine testing

This paper describes a comparison study of test methods and supports the use of real‐time polymerase chain reaction (PCR) for the detection of Clavibacter michiganensis subsp. sepedonicus and Ralstonia solanacearum in potato tubers in routine testing. These 2 bacteria are quarantine organisms under European Union (EU) regulatory control and testing for (latent) infections of these bacteria in seed potatoes is mandatory. Real‐time PCR tests were performed on 276 routine potato tuber samples, including samples infected with either C. michiganensis subsp. sepedonicus or R. solanacearum, and the performance of these real‐time PCR tests was compared with that of immunofluorescence (IF). Real‐time PCR tests, using different primer sets and extraction and PCR protocols, proved to be sensitive and specific for the detection of C. michiganensis subsp. sepedonicus and R. solanacearum in potato tubers in routine testing, and performed at least as well as IF. Real‐time PCR is a good addition to the detection protocols as laid down in EU regulations (EU Council Directives 2006/56/EC and 2006/63/EC).     

Euphresco inter‐laboratory comparison (2009–2012) on detection of Clavibacter michiganensis subsp. sepedonicus and Ralstonia solanacearum in potato tubers: proposal to include TaqMan® real‐time PCR as a primary (core) screening test in EU/EPPO standard methods

In the European Union (EU) potato production is surveyed for Clavibacter michiganensis subsp. sepedonicus (potato ring rot) and Ralstonia solanacearum (potato brown rot) under Commission Directives 93/85/EEC with its amendment 2006/56/EC and 98/57/EEC with its amendment 2006/63/EC. A regular update of the Directives is required in view of developments in understanding of the biology of these organisms and the diagnostics recommended for their detection and identification. Three inter‐laboratory tests (ILT1, ILT2 and ILT3) were performed from 2009 to 2012 as part of a Euphresco Phytosanitary ERA‐NET project to assess performance of current official methods for C. michiganensis subsp. sepedonicus and R. solanacearum. A major aim of the ILTs was to generate data on the performance of real‐time PCR protocols to support their introduction as primary (core) screening tests for both pathogens. In ILT1, 29 laboratories from 23 countries participated, in ILT2, 23 laboratories from 18 countries and in ILT3 42 laboratories from 24 countries. Relative accuracies for real‐time PCR tests averaged 92% for R. solanacearum and 96% for C. michiganensis subsp. sepedonicus) and compared with existing primary (core) screening tests (immunofluorescence, conventional PCR, semi‐selective plating and bioassay) in terms of analytical sensitivity, analytical specificity and robustness. It was concluded that all methods tested, including real‐time PCR, can be considered as equivalent. Therefore TaqMan ^® real‐time PCR is recommended for inclusion in EU Directives and EPPO Standards as a reliable primary (core) screening method.     

玉米细菌性枯萎病菌的环介导恒温扩增(LAMP)检测方法

为了提高口岸和基层实验室检疫和监测玉米细菌性枯萎病菌的准确性和工作效率,利用环介导恒温扩增技术(LAMP),根据内切葡聚糖酶(EGase)基因前导序列,设计2个内引物和2个外引物,对玉米细菌性枯萎病菌进行快速检测。结果表明,使用玉米细菌性枯萎病菌的近缘种或引致相似症状的病原菌菊欧文氏菌玉米致病变种Erwinia chrysanthemi pv.zeae、玉米内州萎蔫病菌Clavibacter michiganensis subsp.nebraskensis、燕麦假单胞菌Pseudomonas avenae、杓兰欧文氏菌Erwinia cypripedii检测其特异性,仅玉米细菌性枯萎病菌有扩增。LAMP检测灵敏度达到2 pg DNA,为普通PCR的100倍;与其它检测方法相比,LAMP方法检测时间短,效率高,不仅降低了设备投入,易于操作,而且具有较高的灵敏度和特异性,适合玉米细菌性枯萎病菌的现场检疫和大规模检测。     

Pathogenicity and phylogenetic analysis of Clavibacter michiganensis strains associated with tomato plants in Iran

During 2013–2016, 277 tomato fields were surveyed across Iran to monitor the status of bacterial canker of tomato, caused by Clavibacter michiganensis subsp. michiganensis. Altogether, 450 plant samples were collected, both with and without symptoms, from which 35 bacterial strains were recovered. These were positive for the PCR test performed using the Clavibacter‐specific primer pair CMR16F1/CMR16R1. Based on the phylogeny of the gyrB gene sequences, 31, three and one of the 35 strains were identified as C. michiganensis, Microbacterium sp. and Agrococcus sp., respectively. The 31 strains of C. michiganensis were further identified as C. michiganensis subsp. michiganensis (23 strains), C. michiganensis subsp. tessellarius (six strains) and Clavibacter spp. (two strains). This was subsequently confirmed by multilocus sequence analysis (MLSA) of five housekeeping genes (atpD, gyrB, ppk, recA and rpoB). In pathogenicity tests, all 23 strains induced wilting symptoms on tomato plants in greenhouse conditions, while no symptoms were observed on eggplant, bell pepper and chili pepper plants. All evaluated pathogenicity determinant genes (celA, pat‐1, tomA, ppaA, chpC and chpG) were detected in 18 out of 31 C. michiganensis strains, using eight specific primer pairs. Estimation of the number of nucleotide differences, sequence similarity matrix and MLSA clustered two peach‐coloured strains (Tom495 and Tom532) separately from all nine previously described subspecies, thereby suggesting these two strains are a new subspecies of C. michiganensis. However, a detailed taxonomic study using multiphased molecular approaches is needed to delineate a formal taxonomic name for these atypical strains.     

Identification and Characterisation of Bacteria Causing Soft-rot in Agave tequilana

Agave tequilana is the raw material for the production of the alcoholic beverage tequila. A bacterial disease has affected the A. tequilana crop in recent years. Previous reports based on colony and cell morphology, Gram stain and potato rot indicated that Erwinia sp. is the main pathogen. We isolated a several bacterial isolates capable of producing soft-rot symptoms in greenhouse pathogenicity assays. An extensive characterisation involving pathogenicity tests, fatty acid profile, metabolic and physiological properties, ribosomal DNA sequence and intergenic transcribed spacer amplification (ITS-PCR) and restriction banding pattern (ITS-RFLP) was made of each isolate. Three different species: Erwinia cacticida, Pantoea agglomerans and Pseudomonas sp. were identified. Fatty acid and metabolic profiles gave low similarity values of identification but 16S rDNA sequence, ITS-PCR and ITS-RFLP confirmed the identification of E. cacticida. In the phylogenetic tree, E. cacticida from blue agave was grouped neither with E. cacticida type strains nor with Erwinia carotovora. This is the first report that associates E. cacticida with A. tequilana soft-rot symptoms.     

Bacterial diseases of potatoes in the major potato-growing areas in Turkey1

In 1988 and 1989 bacterial diseases of potatoes in Central Anatolia were investigated by utilizing the EC method for the detection of ring-rot bacterium (Clavibacter michiganensis ssp. sepedonicus) and using selective medium. 91 samples containing 200 tubers from commercial and farmers’ stores in Central Anatolia were tested for the presence of C.m.sepedonicus and about 50 tubers of each sample were also tested for the presence of soft-rot erwinias. Five different bacteria that cross-reacted with C.m.spedonicus antisera were isolated from heel-end extracts. All cross-reacted bacteria differed from C.m.sepedonicus morphologically and none of them was pathogenic on eggplant. C.m. sepedonicus was not detected, but Erwinia carotouora ssp. carotouora and Erwinia carotovora ssp. atroseptica were present in 7 and 17% of the samples respectively.     

Genetic relationships among strains of <Emphasis Type="Italic">Xanthomonas campestris</Emphasis>pv. <Emphasis Type="Italic">campestris</Emphasis>revealed by novel rep-PCR primers

Novel primers for rep-PCR were developed with the original software and based on `ancient diverged periodical sequences'. Rep-PCR with these primers was applied to study genetic relationships among 51 Xanthomonas campestris strains. The strains were collected from different countries including Russia, Japan, UK, Germany and Hungary. Reference strains of three X. campestrispathovars and five other Xanthomonas species were included. Based on qualitative differences in amplification profiles, the strains were divided into four major groups. Two subgroups recognised within X. campestrispopulation were similar to RFLP haplotypes. The third subgroup included strains of two other pathovariants and Japanese isolates of X. campestris pv. campestriswhile the fourth group comprised the other species of Xanthomonas. The analysis of the diversity within X. campestris resulted in the conclusion that isolates belong to distinct clonal populations (subgroups). The differences between the subgroups of X. campestris were only slightly smaller than between species of Xanthomonas. A PCR fragment about 600 bp amplified by primer KRPN2 was found in nearly all tested strains of X. campestris.SCAR primers designed for this marker produced a single specific band for strains of X. campestris, but not for other Xanthomonas, Pseudomonas and Erwiniastrains tested. Application of the new primer set for rep-PCR offers a rapid, simple and reproducible method for identification of bacterial strains. The X. campestris-specific SCAR primers may be used in diagnostics of this important plant pathogen.     

National control measures for Clavibacter michiganensis subsp. sepedonicus1

Clavibacter michiganensis subsp. sepedonicus has a clearly restricted geographical distribution in the EPPO region, being present almost exclusively in the north and east. Countries where it is absent take strong phytosanitary measures to prevent its introduction. Within the European Union, countries where it is present aim to eradicate the disease. The EU Control Directive for ring rot puts in place community-wide measures for surveillance, containment and eradication.