An Efficient Triplex TaqMan Quantitative PCR to Detect a Blackleg-Causing Lineage of Pectobacterium brasiliense in Potato Based on a Pangenome Analysis.

P. brasiliense is an important bacterial pathogen causing blackleg (BL) in potatoes. Nevertheless, P. brasiliense is often detected in seed lots that do not develop any of the typical blackleg symptoms in the potato crop when planted. Field bioassays identified that P. brasiliense strains can be categorized into two distinct classes, some able to cause blackleg symptoms and some unable to do it. A comparative pangenomic approach was performed on 116 P. brasiliense strains, of which 15 were characterized as BL-causing strains and 25 as non-causative. In a genetically homogeneous clade comprising all BL-causing P. brasiliense strains, two genes only present in the BL-causing strains were identified, one encoding a predicted lysozyme inhibitor Lprl (LZI) and one encoding a putative Toll/interleukin-1 receptor (TIR) domain-containing protein. TaqMan assays for the specific detection of BL-causing P. brasiliense were developed and integrated with the previously developed generic P. brasiliense assay into a triplex TaqMan assay. This simultaneous detection makes the scoring more efficient as only a single tube is needed, and it is more robust as BL-causing strains of P. brasiliense should be positive for all three assays. Individual P. brasiliense strains were found to be either positive for all three assays or only for the P. brasiliense assay. In potato samples, the mixed presence of BL-causing and not BL-causing P. brasiliense strains was observed as shown by the difference in Ct value of the TaqMan assays. However, upon extension of the number of strains, it became clear that in recent years additional BL-causing lineages of P. brasiliense were detected for which additional assays must be developed.

QMRA of Ralstonia solanacearum in potato cultivation: Risks associated with irrigation water recycled through managed aquifer recharge.

Agricultural aquifer storage recovery and transfer (ASTR) stores excess fresh water for later reuse in irrigation. Moreover, water quality improves because chemical pollutants and pathogens will be removed by degradation and attachment to the aquifer material. The source water may contain the bacterial plant pathogen Ralstonia solanacearum which causes plant infections and high yield losses. We used quantitative microbial risk assessment (QMRA) to investigate the removal of R. solanacearum during ASTR to predict infection risks of potato plants after irrigation with the recovered water. Laboratory experiments analyzed the ASTR treatment by investigating the bacterial die-off in the water phase and the removal by attachment to the aquifer sediment. Die-off in the water phase depends on the residence time and ranged between 1.3 and 2.7 log10 after 10 or 60 days water storage, respectively. A subpopulation of the bacteria persisted for a prolonged time at low concentrations which may pose a risk if the water is recovered too early. However, the natural aquifer sand filtration proofed to be highly effective in removing R. solanacearum by attachment which depends on the distance between injection and abstraction well. The high removal by attachment alone (18 log10 after 1 m) would reduce bacterial concentrations to negligible numbers. Upscaling to longer soil passages is discussed in the paper. Infection risks of potato plants were calculated using a dose-response model and ASTR treatment resulted in negligible infection risks of a single plant, but also when simulating the irrigation of a 5 ha potato field. This is the first QMRA that analyzed an agricultural ASTR and the fate of a plant pathogen focusing on plant health. QMRA is a useful (water) management tool to evaluate the treatment steps of water reclamation technologies with the aim to provide safe irrigation water and reduce risks disseminating plant diseases.

Inactivation of tomato WAT1 leads to reduced susceptibility to Clavibacter michiganensis through downregulation of bacterial virulence factors.

Tomato bacterial canker caused by Clavibacter michiganensis (Cm) is considered to be one of the most destructive bacterial diseases of tomato. To date, no resistance to the pathogen has been identified. While several molecular studies have identified (Cm) bacterial factors involved in disease development, the plant genes and mechanisms associated with susceptibility of tomato to the bacterium remain largely unknown. Here, we show for the first time that tomato gene SlWAT1 is a susceptibility gene to Cm. We inactivated the gene SlWAT1 through RNAi and CRISPR/Cas9 to study changes in tomato susceptibility to Cm. Furthermore, we analysed the role of the gene in the molecular interaction with the pathogen. Our findings demonstrate that SlWAT1 functions as an S gene to genetically diverse Cm strains. Inactivation of SlWAT1 reduced free auxin contents and ethylene synthesis in tomato stems and suppressed the expression of specific bacterial virulence factors. However, CRISPR/Cas9 slwat1 mutants exhibited severe growth defects. The observed reduced susceptibility is possibly a result of downregulation of bacterial virulence factors and reduced auxin contents in transgenic plants. This shows that inactivation of an S gene may affect the expression of bacterial virulence factors.

Removal of bacterial plant pathogens in columns filled with quartz and natural sediments under anoxic and oxygenated conditions.

Irrigation with surface water carrying plant pathogens poses a risk for agriculture. Managed aquifer recharge enhances fresh water availability while simultaneously it may reduce the risk of plant diseases by removal of pathogens during aquifer passage. We compared the transport of three plant pathogenic bacteria with Escherichia coli WR1 as reference strain in saturated laboratory column experiments filled with quartz sand, or sandy aquifer sediments. E. coli showed the highest removal, followed by Pectobacterium carotovorum, Dickeya solani and Ralstonia solanacearum. Bacterial and non-reactive tracer breakthrough curves were fitted with Hydrus-1D and compared with colloid filtration theory (CFT). Bacterial attachment to fine and medium aquifer sand under anoxic conditions was highest with attachment rates of max. katt1 = 765 day-1 and 355 day-1, respectively. Attachment was the least to quartz sand under oxic conditions (katt1 = 61 day-1). In CFT, sticking efficiencies were higher in aquifer than in quartz sand but there was no differentiation between fine and medium aquifer sand. Overall removal ranged between < 6.8 log10 m-1 in quartz and up to 40 log10 m-1 in fine aquifer sand. Oxygenation of the anoxic aquifer sediments for two weeks with oxic influent water decreased the removal. The results highlight the potential of natural sand filtration to sufficiently remove plant pathogenic bacteria during aquifer storage.

The Broad Host Range Plant Pathogen Dickeya dianthicola Shows a High Genetic Diversity.

The wide host range phytopathogen D. dianthicola, first described in ornamentals in the 1950s, rapidly became a threat for potato production in Europe and, more recently, worldwide. Previous genomic analyses, mainly of strains isolated from potato, revealed little sequence diversity. To further analyse D. dianthicola genomic diversity, we used a larger genome panel of 41 isolates encompassing more strains isolated from potato over a wide time scale and more strains isolated from other hosts. The phylogenetic and pan-genomic trees revealed a large cluster of highly related genomes but also the divergence of two more distant strains, IPO 256 and 67.19, isolated from potato and impatiens, respectively, and the clustering of the three strains isolated from Kalanchoe with one more distinct potato strain. An SNP-based minimal spanning tree highlighted both diverse clusters of (nearly) clonal strains and several strains scattered in the MST, irrespective of country or date of isolation, that differ by several thousand SNPs. This study reveals a higher diversity in D. dianthicola than previously described. It indicates the clonal spread of this pathogen over long distances, as suspected from worldwide seed trading, and possible multiple introductions of D. dianthicola from alternative sources of contaminations.

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

BACKGROUND: Bacterial 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 SYMPTOMS: On 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 RANGE: Potato is the main host of the pathogen, but natural infection also occurs on eggplant, tomato, and sugar beet. TAXONOMIC STATUS OF THE PATHOGEN: Class: 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 PROPERTIES: Gram-positive, club-shaped cells with creamy to yellowish-cream colonies for which the optimal growth temperature is 20-23°C. DISTRIBUTION: Asia (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 CATEGORIZATION: CORBSE: EPPO A2 list no. 51. EU; Annex designation I/A2.

Dose-response relationship of Ralstonia solanacearum and potato in greenhouse and in vitro experiments.

Ralstonia solanacearum is the causative agent of bacterial wilt of potato and other vegetable crops. Contaminated irrigation water contributes to the dissemination of this pathogen but the exact concentration or biological threshold to cause an infection is unknown. In two greenhouse experiments, potted potato plants (Solanum tuberosum) were exposed to a single irrigation with 50 mL water (non-invasive soil-soak inoculation) containing no or 102 - 108 CFU/mL R. solanacearum. The disease response of two cultivars, Kondor and HB, were compared. Disease development was monitored over a three-month period after which stems, roots and tubers of asymptomatic plants were analyzed for latent infections. First wilting symptoms were observed 15 days post inoculation in a plant inoculated with 5x109 CFU and a mean disease index was used to monitor disease development over time. An inoculum of 5x105 CFU per pot (1.3x102 CFU/g soil) was the minimum dose required to cause wilting symptoms, while one latent infection was detected at the lowest dose of 5x102 CFU per pot (0.13 CFU/g). In a second set of experiments, stem-inoculated potato plants grown in vitro were used to investigate the dose-response relationship under optimal conditions for pathogen growth and disease development. Plants were inoculated with doses between 0.5 and 5x105 CFU/plant which resulted in visible symptoms at all doses. The results led to a dose-response model describing the relationship between R. solanacearum exposure and probability of infection or illness of potato plants. Cultivar Kondor was more susceptible to brown-rot infections than HB in greenhouse experiments while there was no significant difference between the dose-response models of both cultivars in in vitro experiments. The ED50 for infection of cv Kondor was 1.1x107 CFU. Results can be used in management strategies aimed to reduce or eliminate the risk of bacterial wilt infection when using treated water in irrigation.

Susceptibility reversed: modified plant susceptibility genes for resistance to bacteria.

Plants have evolved complex defence mechanisms to avoid invasion of potential pathogens. Despite this, adapted pathogens deploy effector proteins to manipulate host susceptibility (S) genes, rendering plant defences ineffective. The identification and mutation of plant S genes exploited by bacterial pathogens are important for the generation of crops with durable and broad-spectrum resistance. Application of mutant S genes in the breeding of resistant crops is limited because of potential pleiotropy. New genome editing techniques open up new possibilities for the modification of S genes. In this review, we focus on S genes manipulated by bacteria and propose ways for their identification and precise modification. Finally, we propose that genes coding for transporter proteins represent a new group of S genes.

Die-off of plant pathogenic bacteria in tile drainage and anoxic water from a managed aquifer recharge site.

Managed aquifer recharge (MAR) can provide irrigation water and overcome water scarcity in agriculture. Removal of potentially present plant pathogens during MAR is essential to prevent crop diseases. We studied the die-off of three plant pathogenic bacteria in water microcosms with natural or filtered tile drainage water (TDW) at 10 and 25°C and with natural anoxic aquifer water (AW) at 10°C from a MAR site. These bacteria were: Ralstonia solanacearum (bacterial wilt), and the soft rot Pectobacteriaceae (SRP) Dickeya solani and Pectobacterium carotovorum sp. carotovorum (soft rot, blackleg). They are present in surface waters and cause destructive crop diseases worldwide which have been linked to contaminated irrigation water. Nevertheless, little is known about the survival of the SRP in aqueous environments and no study has investigated the persistence of R. solanacearum under natural anoxic conditions. We found that all bacteria were undetectable in 0.1 mL samples within 19 days under oxic conditions in natural TDW at 10°C, using viable cell counting, corresponding to 3-log10 reduction by die-off. The SRP were no longer detected within 6 days at 25°C, whereas R. solanacearum was detectable for 25 days. Whereas in anoxic natural aquifer water at 10°C, the bacterial concentrations declined slower and the detection limit was reached within 56 days. Finally, we modelled the inactivation curves with a modified Weibull model that can simulate different curve shapes such as shoulder phenomena in the beginning and long tails reflecting persistent bacterial populations. The non-linear model was shown to be a reliable tool to predict the die-off of the analysed plant pathogenic bacteria, suggesting its further application to other pathogenic microorganisms in the context of microbial risk assessment.

The Pectobacterium pangenome, with a focus on Pectobacterium brasiliense, shows a robust core and extensive exchange of genes from a shared gene pool.

BACKGROUND: Bacterial plant pathogens of the Pectobacterium genus are responsible for a wide spectrum of diseases in plants, including important crops such as potato, tomato, lettuce, and banana. Investigation of the genetic diversity underlying virulence and host specificity can be performed at genome level by using a comprehensive comparative approach called pangenomics. A pangenomic approach, using newly developed functionalities in PanTools, was applied to analyze the complex phylogeny of the Pectobacterium genus. We specifically used the pangenome to investigate genetic differences between virulent and avirulent strains of P. brasiliense, a potato blackleg causing species dominantly present in Western Europe. RESULTS: Here we generated a multilevel pangenome for Pectobacterium, comprising 197 strains across 19 species, including type strains, with a focus on P. brasiliense. The extensive phylogenetic analysis of the Pectobacterium genus showed robust distinct clades, with most detail provided by 452,388 parsimony-informative single-nucleotide polymorphisms identified in single-copy orthologs. The average Pectobacterium genome consists of 47% core genes, 1% unique genes, and 52% accessory genes. Using the pangenome, we zoomed in on differences between virulent and avirulent P. brasiliense strains and identified 86 genes associated to virulent strains. We found that the organization of genes is highly structured and linked with gene conservation, function, and transcriptional orientation. CONCLUSION: The pangenome analysis demonstrates that evolution in Pectobacteria is a highly dynamic process, including gene acquisitions partly in clusters, genome rearrangements, and loss of genes. Pectobacterium species are typically not characterized by a set of species-specific genes, but instead present themselves using new gene combinations from the shared gene pool. A multilevel pangenomic approach, fusing DNA, protein, biological function, taxonomic group, and phenotypes, facilitates studies in a flexible taxonomic context.

Transcriptome analysis of Xanthomonas fragariae in strawberry leaves.

Xanthomonas fragariae is a quarantine bacterial pathogen that causes angular leaf spot on strawberry. The aim of our study was to analyse the mechanism of interaction of this bacterium with its host plant at the transcriptome level. For this purpose, mRNAs of X. fragariae growing in Wilbrink's medium and from infected strawberry cv. Elsanta plants were isolated and sequenced using the Illumina MiSeq platform. The expression profiles of the bacteria in Wilbrink's medium and in planta were very diverse. Of the 3939 CDSs recorded, 1995 had significantly different expression in planta (966 and 1029 genes were down- and upregulated, respectively). Among the genes showing increased expression in planta, those with eggNOG/COG (evolutionary genealogy of genes: Non-supervised Orthologous Groups/Cluster of Orthologous Groups) categories associated with bacterial cell motility, signal transduction, transport and metabolism of inorganic ions and carbohydrates and transcription were overrepresented. Among the genes with the most increased expression in planta, genes primarily associated with flagella synthesis and chemotaxis were found. It is also interesting to note that out of the 31 genes localized on a plasmid, 16 were expressed differently in planta, which may indicate their potential role in plant-pathogen interactions. Many genes with differentiated expression that were localized on chromosome and plasmid encode proteins of unknown function.

Potato-Infecting Ralstonia solanacearum Strains in Iran Expand Knowledge on the Global Diversity of Brown Rot Ecotype of the Pathogen.

Bacterial wilt and brown rot disease caused by Ralstonia solanacearum species complex (RSSC) is one of the major constraints of potato (Solanum tuberosum) production around the globe. During 2017 to 2018, an extensive field survey was conducted in six potato-growing provinces of Iran to monitor the status of bacterial wilt disease. Pathogenicity and host range assays using 59 bacterial strains isolated in Iran showed that they were pathogenic on eggplant, red nightshade, pepper, potato and tomato, while nonpathogenic on common bean, cowpea, cucumber, sunflower, zinnia and zucchini. PCR-based diagnosis revealed that the strains belong to the phylotype IIB/sequevar 1 (IIB/I) lineage of the RSSC. Furthermore, a five-gene multilocus sequence analysis and typing (egl, fliC, gyrB, mutS, and rplB) confirmed the phylogenetically near-homogeneous nature of the strains within IIB/I lineage. Four sequence types were identified among 58 IIB/1 strains isolated in Iran. Phylogenetically near-homogeneous nature of the strains in Iran raise questions about the mode of inoculum entry of the bacterial wilt pathogen into the country (one-time introduction versus multiple introductions), while the geographic origin of the Iranian R. solanacearum strains remains undetermined. Furthermore, sequence typing showed that there were shared alleles (haplotypes) and sequence types among the strains isolated in geographically distant areas in Iran, suggesting intranational transmission of the pathogen in the country.

Genome Resource of Two Potato Strains of Ralstonia solanacearum Biovar 2 (Phylotype IIB Sequevar 1) and Biovar 2T (Phylotype IIB Sequevar 25) Isolated from Lowlands in Iran.

Ralstonia solanacearum, the causal agent of bacterial wilt and brown rot disease, is one of the major pathogens of solanaceous crops, including potato, around the globe. Biovar 2T (phylotype II/sequevar 25) of R. solanacearum is adapted to tropical lowlands and is only reported in South America and Iran. Thus far, no genome resource of the biovar 2T of the pathogen has been available. Here, we present the near-complete genome sequences of the biovar 2T strain CFBP 8697 as well as strain CFBP 8695 belonging to biovar 2 race 3, both isolated from potato in Iran. The genomic data of biovar 2T will extend our understanding of the virulence features of R. solanacearum and pave the way for research on biovar 2T functional and interaction genetics.

Potato Virus Y Detection in Seed Potatoes Using Deep Learning on Hyperspectral Images.

Virus diseases are of high concern in the cultivation of seed potatoes. Once found in the field, virus diseased plants lead to declassification or even rejection of the seed lots resulting in a financial loss. Farmers put in a lot of effort to detect diseased plants and remove virus-diseased plants from the field. Nevertheless, dependent on the cultivar, virus diseased plants can be missed during visual observations in particular in an early stage of cultivation. Therefore, there is a need for fast and objective disease detection. Early detection of diseased plants with modern vision techniques can significantly reduce costs. Laboratory experiments in previous years showed that hyperspectral imaging clearly could distinguish healthy from virus infected potato plants. This paper reports on our first real field experiment. A new imaging setup was designed, consisting of a hyperspectral line-scan camera. Hyperspectral images were taken in the field with a line interval of 5 mm. A fully convolutional neural network was adapted for hyperspectral images and trained on two experimental rows in the field. The trained network was validated on two other rows, with different potato cultivars. For three of the four row/date combinations the precision and recall compared to conventional disease assessment exceeded 0.78 and 0.88, respectively. This proves the suitability of this method for real world disease detection.

Genome-based population structure analysis of the strawberry plant pathogen Xanthomonas fragariae reveals two distinct groups that evolved independently before its species description.

Xanthomonas fragariae is a quarantine organism in Europe, causing angular leaf spots on strawberry plants. It is spreading worldwide in strawberry-producing regions due to import of plant material through trade and human activities. In order to resolve the population structure at the strain level, we have employed high-resolution molecular typing tools on a comprehensive strain collection representing global and temporal distribution of the pathogen. Clustered regularly interspaced short palindromic repeat regions (CRISPRs) and variable number of tandem repeats (VNTRs) were identified within the reference genome of X. fragariae LMG 25863 as a potential source of variation. Strains from our collection were whole-genome sequenced and used in order to identify variable spacers and repeats for discriminative purpose. CRISPR spacer analysis and multiple-locus VNTR analysis (MLVA) displayed a congruent population structure, in which two major groups and a total of four subgroups were revealed. The two main groups were genetically separated before the first X. fragariae isolate was described and are potentially responsible for the worldwide expansion of the bacterial disease. Three primer sets were designed for discriminating CRISPR-associated markers in order to streamline group determination of novel isolates. Overall, this study describes typing methods to discriminate strains and monitor the pathogen population structure, more especially in the view of a new outbreak of the pathogen.

Genetic Characterization of Clavibacter michiganensis subsp. michiganensis Population in Turkey.

The pathogenic gram-positive bacterium Clavibacter michiganensis subsp. michiganensis (Smith) Davis et al. is the most harmful bacterium to tomatoes in many countries with a cooler climate. Multilocus sequence analysis was performed on five housekeeping genes (bipA, gyrB, kdpA, ligA, and sdhA) and three virulence-related genes (ppaA, chpC, and tomA) to determine evolutionary relationships and population structure of 108 C. michiganensis subsp. michiganensis strains collected from Turkey between 1996 and 2012. Based on these analyses, we concluded that C. michiganensis subsp. michiganensis in Turkey is highly uniform. However, at least four novel C. michiganensis subsp. michiganensis strains were recently introduced, possibly at the beginning of the 1990s. The singletons might point to additional sources or to strains that have evolved locally in Turkey.

Complete Genome Sequences of Three Isolates of Xanthomonas fragariae, the Bacterium Responsible for Angular Leaf Spots on Strawberry Plants.

Xanthomonas fragariae is a worldwide-spread plant bacterial disease causing angular leaf spots, thus reducing the yield of production for strawberry fruits. Three isolates with various geographic and time origins were sequenced with long-read technology (PacBio) to generate finished genome sequences of virulent strains and observe the variability in their contents.

Dickeya solani sp. nov., a pectinolytic plant-pathogenic bacterium isolated from potato (Solanum tuberosum).

Pectinolytic bacteria have been recently isolated from diseased potato plants exhibiting blackleg and slow wilt symptoms found in a number of European countries and Israel. These Gram-reaction-negative, motile, rods were identified as belonging to the genus Dickeya, previously the Pectobacterium chrysanthemi complex (Erwinia chrysanthemi), on the basis of production of a PCR product with the pelADE primers, 16S rRNA gene sequence analysis, fatty acid methyl esterase analysis, the production of phosphatases and the ability to produce indole and acids from α-methylglucoside. Differential physiological assays used previously to differentiate between strains of E. chrysanthemi, showed that these isolates belonged to biovar 3. Eight of the isolates, seven from potato and one from hyacinth, were analysed together with 21 reference strains representing all currently recognized taxa within the genus Dickeya. The novel isolates formed a distinct genetic clade in multilocus sequence analysis (MLSA) using concatenated sequences of the intergenic spacer (IGS), as well as dnaX, recA, dnaN, fusA, gapA, purA, rplB, rpoS and gyrA. Characterization by whole-cell MALDI-TOF mass spectrometry, pulsed field gel electrophoresis after digestion of whole-genome DNA with rare-cutting restriction enzymes, average nucleotide identity analysis and DNA-DNA hybridization studies, showed that although related to Dickeya dadantii, these isolates represent a novel species within the genus Dickeya, for which the name Dickeya solani sp. nov. (type strain IPO 2222(T) = LMG25993(T) = NCPPB4479(T)) is proposed.

Draft genome sequence of the biocontrol strain Serratia plymuthica A30, isolated from rotting potato tuber tissue.

Serratia plymuthica A30 is a Gram-negative bacterium expressing antagonistic activity toward blackleg- and soft rot-causing Dickeya sp. biovar 3 ("Dickeya solani"). Here, we present the draft genome sequence of strain A30, which has been isolated from rotten potato tuber tissue.

Quantitative and specific detection of the biocontrol agent, Serratia plymuthica, in plant extracts using a real-time TaqMan® assay.

A Serratia plymuthica-specific TaqMan® assay was designed based on the consensus nucleotide sequence from the 3'- end of the luxS gene present in all S. plymuthica strains tested. The specificity of the assay was demonstrated by testing 21 Serratia spp. strains and 30 isolates belonging to various species that can potentially coexist with S. plymuthica in the same environment. Positive reactions in the TaqMan® assay were observed only for S. plymuthica isolates and not for other bacteria. The TaqMan® assay could detect down to 1.95 ng of S. plymuthica DNA, down to 5 bacterial cells per reaction (100 cfu ml(-1)) in vitro, down to 50 bacterial cells per reaction (1,000 cfu ml(-1)) in spiked potato root extracts and down to 5 bacterial cells per reaction (100 cfu ml(-1)) in spiked potato haulm extracts. We used this assay to quantify S. plymuthica A30 cells in potato and tomato haulms and roots grown from S. plymuthica A30-inoculated potato seed tubers and tomato seeds. The results were comparable with the spread-plating of plant extracts on a newly developed S. plymuthica A30 selective medium (CVTR2Arif). The TaqMan® assay can be used to quantify S. plymuthica isolates in different ecosystems and in complex substrates.