Symptoms and yield reduction caused by Dickeya spp. strains isolated from potato and river water in Finland

Biochemical characterisation of Dickeya strains isolated from potato plants and river water samples in Finland showed that the majority of the strains were biovar 3. They thus resembled the strains recently isolated from potato in the Netherlands, Poland and Israel and form a new clade within the Dickeya genus. About half of the Finnish isolates resembling strains within this new clade were virulent and caused wilting, necrotic lesions and rotting of leaves and stems. Similar symptoms were caused by D. dianthicola strains isolated from one potato sample and from several river water samples. Frequently, the rotting caused by the Dickeya strains was visible in the upper parts of the stem, while the stem base was necrotic from the pith but hard and green on the outside, resulting in symptoms quite different from the blackleg caused by Pectobacterium atrosepticum. The presence of Dickeya in the symptomatic plants in the field assay was verified with a conventional PCR and with a real-time PCR test developed for the purpose. The virulent Dickeya strains reduced the yield of individual plants by up to 50% and caused rotting of the daughter tubers in the field and in storage. Management of Dickeya spp. in the potato production chain requires awareness of the symptoms and extensive knowledge about the epidemiology of the disease.     

Biochemical and genetical analysis reveal a new clade of biovar 3 <Emphasis Type="Italic">Dickeya</Emphasis> spp. strains isolated from potato in Europe

Sixty-five potato strains of the soft rot-causing plant pathogenic bacterium Dickeya spp., and two strains from hyacinth, were characterised using biochemical assays, REP-PCR genomic finger printing, 16S rDNA and dnaX sequence analysis. These methods were compared with nineteen strains representing six Dickeya species which included the type strains. A group of twenty-two potato strains isolated between 2005-2007 in the Netherlands, Poland, Finland and Israel were characterised as belonging to biovar 3. They were 100% identical in REP-PCR, dnaX and 16S rDNA sequence analysis. In a polyphasic analysis they formed a new clade different from the six Dickeya species previously described, and may therefore constitute a new species. The strains were very similar to a Dutch strain from hyacinth. On the basis of dnaX sequences and biochemical assays, all other potato strains isolated in Europe between 1979 and 1994 were identified as D. dianthicola (biovar 1 and 7), with the exception of two German strains classified as D. dieffenbachia (biovar 2) and D. dadantii (biovar 3), respectively. Potato strains from Peru were classified as D. dadantii, from Australia as D. zeae and from Taiwan as D. chrysanthemi bv. parthenii, indicating that different Dickeya species are found in association with potato.     

<Emphasis Type="Italic">Pectobacterium carotovorum</Emphasis> subsp. <Emphasis Type="Italic">carotovorum</Emphasis> can cause potato blackleg in temperate climates

It is well established that the pectinolytic bacteria Pectobacterium atrosepticum (Pca) and Dickeya spp. are causal organisms of blackleg in potato. In temperate climates, the role of Pectobacterium carotovorum subsp. carotovorum (Pcc) in potato blackleg, however, is unclear. In different western and central European countries plants are frequently found with blackleg from which only Pcc can be isolated, but not Pca or Dickeya spp. Nevertheless, tubers vacuum-infiltrated with Pcc strains have so far never yielded blackleg-diseased plants in field experiments in temperate climates. In this study, it is shown that potato tubers, vacuum-infiltrated with a subgroup of Pcc strains isolated in Europe, and planted in two different soil types, can result in up to 50% blackleg diseased plants.     

Genes responsible for coronatine synthesis in <Emphasis Type="Italic">Pseudomonas syringae</Emphasis> present in the genome of soft rot bacteria

Primers for the PCR amplification of homologous genes encoding polyketide coronafacic acid and coronafacic ligase in the cells of Pectobacterium atrosepticum SCRI1043 (BX950851) were developed to study the presence of these genes in the genome of Pectobacterium sp. and Dickeya sp. Coronafacic ligase catalyses the formation of coronatine from polyketide coronafacic acid and coronamic acid. Coronatine is a toxin produced by Pseudomonas syringae and is one of the major virulence factors in this bacterium. This study using several strains of P. atrosepticum, P. carotovorum subsp. carotovorum and Dickeya sp. isolated in different countries, indicated that all strains of P. atrosepticum possess genes coding coronafacic acid (cfa gene cluster) and coronafacic ligase (cfl). However, these genes were present only in the genome of five out of 50 tested P. carotovorum subsp. carotovorum strains and two out of 34 strains of Dickeya sp. tested. The PCR products homologous to the sequence of cfa7 and cfl gene fragments were sequenced in order to check the level of homology between genes of P. atrosepticum, P. carotovorum subsp. carotovorum and Dickeya sp. The sequences of the gene fragments amplified from all P. atrosepticum strains were almost identical (100% and 99.97%, respectively). The homology of the sequences obtained for P. atrosepticum and sequences of five P. carotovorum subsp. carotovorum and two Dickeya sp. was lower, between 89.69% to 95.00% for the cfl gene fragment, and about 94% for the cfa7 gene fragment.     

Assessment of recent outbreaks of <Emphasis Type="Italic">Dickeya</Emphasis> sp. (syn. <Emphasis Type="Italic">Erwinia chrysanthemi</Emphasis>) slow wilt in potato crops in Israel

Suspected Dickeya sp. strains were obtained from potato plants and tubers collected from commercial plots. The disease was observed on crops of various cultivars grown from seed tubers imported from the Netherlands during the spring seasons of 2004–2006, with disease incidence of 2–30% (10% in average). In addition to typical wilting symptoms on the foliage, in cases of severe infection, progeny tubers were rotten in the soil. Six strains were characterised by biochemical, serological and PCR-amplification. All tests verified the strains as Dickeya sp. The rep-PCR and the biochemical assays showed that the strains isolated from blackleg diseased plants in Israel were very similar, if not identical to strains isolated from Dutch seed potatoes, suggesting that the infection in Israel originated from the Dutch seed. The strains were distantly related to D. dianthicola strains, typically found in potatoes in Western Europe, and were similar to biovar 3 D. dadanti or D. zeae. This is the first time that the presence of biovar 3 strains in potato in the Netherlands is described. One of the strains was used for pathogenicity assays on potato cvs Nicola and Mondial. Symptoms appeared 2 to 3 days after stem inoculation, and 7 to 10 days after soil inoculation. The control plants treated with water, or plants inoculated with Pectobacterium carotovorum, did not develop any symptoms with either method of inoculation. The identity of Dickeya sp. and P. carotovorum re-isolated from inoculated plants was confirmed by PCR and ELISA.     

Simultaneous and selective detection of two major soft rot pathogens of potato: Pectobacterium atrosepticum (Erwinia carotovora subsp. atrosepticum) and Dickeya spp. (Erwinia chrysanthemi)

Dickeya spp. and Pectobacterium atrosepticum are major pathogens of potato. Current methods to detect these soft-rotting bacteria require separate identification steps. Here we describe a simple method allowing simultaneous detection of both pathogens based on multiplex PCR. The sensitivity of the primer sets was first examined on purified genomic DNA of the type strains Dickeya chrysanthemi 2048^T and P. atrosepticum 1526^T. The specificity and detection limits of the primer sets were successfully tested on 61 strains belonging to various Dickeya and Pectobacterium species, on artificially inoculated and on naturally contaminated potato plants. This new method provides a gain in time and materials, the main advantages for large-scale processes such as pathogen-free seed certification.     

Biochemical,physiological, and molecular characterization of <Emphasis Type="Italic">Dickeya dianthicola</Emphasis> (formerly named <Emphasis Type="Italic"> Erwinia chrysanthemi</Emphasis>) causing potato blackleg disease in Japan

The taxonomic assignment of Japanese potato blackleg isolates of Dickeya spp. has not been confirmed after the changes in their former name, Erwinia chrysanthemi. Therefore, we investigated and identified 23 representative isolates of Dickeya spp. from symptomatic stems of potatoes in Japan, with biochemical tests and phylogenetic sequence analysis using recA, dnaX, rpoD, gyrB, and 16S rDNA sequences. Results of our biochemical tests showed that all isolates can be assigned to phenon 5 and biovar 1, which are associated with D. dianthicola. Based on the recA, dnaX, rpoD, gyrB, and 16S rDNA sequences, all isolates are in the same clade with D. dianthicola and were clearly distinguished from D. chrysanthemi, D. dadantii, D. dadantii subsp. dieffenbachiae, D. solani, D. zeae, and D. paradisiaca. Therefore, we conclude that Dickeya spp. isolated from potatoes with blackleg symptoms in Japan are D. dianthicola.     

Characterization of <Emphasis Type="Italic">Dickeya</Emphasis> and <Emphasis Type="Italic">Pectobacterium</Emphasis> strains obtained from diseased potato plants in different climatic conditions of Norway and Poland

Soft rot and blackleg of potato caused by pectinolytic bacteria lead to severe economic losses in potato production worldwide. To investigate the species composition of bacteria causing soft rot and black leg of potato in Norway and Poland, bacteria were isolated from potato tubers and stems. Forty-one Norwegian strains and 42 Polish strains that formed cavities on pectate medium were selected for potato tuber maceration assays and sequencing of three housekeeping genes (dnaX, icdA and mdh) for species identification and phylogenetic analysis. The distribution of the species causing soft rot and blackleg in Norway and Poland differed: we have demonstrated that mainly P. atrosepticum and P. c. subsp. carotovorum are the causal agents of soft rot and blackleg of potatoes in Norway, while P. wasabiae was identified as one of the most important soft rot pathogens in Poland. In contrast to the other European countries, D. solani seem not to be a major pathogen of potato in Norway and Poland. The Norwegian and Polish P. c. subsp. carotovorum and P. wasabiae strains did not cluster with type strains of the respective species in the phylogenetic analysis, which underlines the taxonomic complexity of the genus Pectobacterium. No correlation between the country of origin and clustering of the strains was observed. All strains tested in this study were able to macerate potato tissue. The ability to macerate potato tissue was significantly greater for the P. c. subsp. carotovorum and Dickeya spp., compared to P. atrosepticum and P. wasabiae.     

Distribution of <Emphasis Type="Italic">Dickeya</Emphasis> spp. and <Emphasis Type="Italic">Pectobacterium carotovorum</Emphasis> subsp. <Emphasis Type="Italic">carotovorum</Emphasis> in naturally infected seed potatoes

Detailed studies were conducted on the distribution of Pectobacterium carotovorum subsp. carotovorum and Dickeya spp. in two potato seed lots of different cultivars harvested from blackleg-diseased crops. Composite samples of six different tuber sections (peel, stolon end, and peeled potato tissue 0.5, 1.0, 2.0 and 4.0 cm from the stolon end) were analysed by enrichment PCR, and CVP plating followed by colony PCR on the resulting cavity-forming bacteria. Seed lots were contaminated with Dickeya spp. and P. carotovorum subsp. carotovorum (Pcc), but not with P. atrosepticum. Dickeya spp. and Pcc were found at high concentrations in the stolon ends, whereas relatively low densities were found in the peel and in deeper located potato tissue. Rep-PCR, 16S rDNA sequence analysis and biochemical assays, grouped all the Dickeya spp. isolates from the two potato seed lots as biovar 3. The implications of the results for the control of Pectobacterium and Dickeya spp., and sampling strategies in relation to seed testing, are discussed.     

Phenotypic diversity,host range and molecular phylogeny of <Emphasis Type="Italic">Dickeya</Emphasis> isolates from Spain

Six Dickeya spp. strains representative of a larger group of bacteria isolated from potato, onion and irrigation water in Spain between years 2003–2005, were characterised by biochemical, serological, molecular and pathogenicity assays. Biochemical and serological differences, as well as pathogenic behaviour in host range and virulence levels, were observed among the strains. They were classified into biovars 3 and 6. Phylogenetic analysis and comparison of the isolates with type strains of Dickeya species characterised to date were performed using concatenated partial sequences of the housekeeping genes gapA and mdh. One of the Spanish strains was identified as D. dieffenbachiae, whereas the other ones did not fit clearly into the previously described six Dickeya species, and may therefore constitute novel species. Isolation of dissimilar pathogenic strains in different rivers and irrigation water sources supports the idea that Dickeya species is commonly present in such an environment, and contaminated water is a potential source of inoculum for the disease in different crops.     

Dickeya species: an emerging problem for potato production in Europe

Dickeya species (formerly Erwinia chrysanthemi) cause diseases on numerous crop and ornamental plants world‐wide. Dickeya spp. (probably D. dianthicola) were first reported on potato in the Netherlands in the 1970s and have since been detected in many other European countries. However, since 2004–5 a new pathogen, with the proposed name ‘D. solani’, has been spreading across Europe via trade in seed tubers and is causing increasing economic losses. Although disease symptoms are often indistinguishable from those of the more established blackleg pathogen Pectobacterium spp., Dickeya spp. can initiate disease from lower inoculum levels, have a greater ability to spread through the plant’s vascular tissue, are considerably more aggressive, and have higher optimal temperatures for disease development (the latter potentially leading to increased disease problems as Europe’s climate warms). However, they also appear to be less hardy than Pectobacterium spp. in soil and other environments outside the plant. Scotland is currently the only country in Europe to enforce zero tolerance for Dickeya spp. in its potato crop in an attempt to keep its seed tuber industry free from disease. However, there are a number of other ways to control the disease, including seed tuber certification, on‐farm methods and the use of diagnostics. For diagnostics, new genomics‐based approaches are now being employed to develop D. dianthicola‐ and ‘D. solani’‐specific PCR‐based tests for rapid detection and identification. It is hoped that these diagnostics, together with other aspects of ongoing research, will provide invaluable tools and information for controlling this serious threat to potato production.     

Characterization of Dickeya strains isolated from potato grown under hot‐climate conditions

Dickeya strains isolated in Israel in 2006–2010 were characterized by dnaX sequence analysis, pulsed‐field gel electrophoresis (PFGE), biochemical assays and pectolytic activity, and found to be homogeneous: most of them could be classified as ‘Dickeya solani’. Of the 34 strains isolated from imported seed tubers or potato plants grown from imported seed, 32 were typed as ‘D. solani’ and only two were characterized as Dickeya dianthicola. Biovar typing indicated that all ‘D. solani’ strains were biovar 3. ‘Dickeya solani’ strains were most closely related to Dickeya dadantii subsp. dieffenbachiae according to PFGE and dnaX analyses and both species exhibited high pectolytic activity. Expression levels of two putative virulence genes, pelL (encoding a pectic enzyme) and dspE (encoding a type III effector) were significantly induced in ‘D. solani’ strains isolated from potato plants or tubers grown in hot climates such as the Negev region in Israel, compared to those isolated from seed tubers imported from the Netherlands, France or Germany. Results of this study support the hypothesis that ‘D. solani’ strains isolated in Israel are also clonal; however, they appear to be more virulent than strains isolated in Europe.     

The effect of temperature on the phenotypic features and the maceration ability of <Emphasis Type="Italic">Dickeya solani</Emphasis> strains isolated in Finland,Israel and Poland

Pectinolytic bacteria from the genus Dickeya (former Erwinia chrysanthemi), belonging to Dickeya dianthicola and Dickeya solani species, are causative agents of blackleg and soft rot diseases in Europe. Recently, D. solani have been isolated most frequently from potato plants with the symptoms of blackleg and soft rot. D. solani strains were shown to cause more severe disease symptoms on potato plants than D. dianthicola especially at the higher temperature. They are also able to develop blackleg disease from lower inoculum levels. In the presented study we not only compared phenotypic features of fifteen D. solani strains isolated in countries having different climatic conditions, Poland, Finland and Israel, but also we examined three D. dianthicola strains. The comparison was performed to determine the influence of the strain origin and the temperature of incubation on the ability of the strains to macerate potato tissue and on their major virulence factors such as: pectinolytic, cellulolytic and proteolytic activities, siderophore production and motility. Polish D. solani strains showed higher activities of cell wall degrading enzymes than the Finnish and Israeli strains at all the tested temperatures: 18, 27, 37 °C. This observation is correlated with the higher ability of Polish D. solani strains to cause soft rot. In addition, D. solani strains exhibited higher activity of the above mentioned enzymes and caused more severe potato tuber maceration in laboratory tests than the tested D. dianthicola strains. The collected results indicate that although D. solani strains from different climatic conditions have identical Pulse Field Gel Electrophoresis (PFGE) profiles in addition to the same fingerprint profiles obtained by the repetitive sequence-based polymerase chain reaction (REP, ERIC and BOX repetitive sequences), they differ in the examined phenotypic features, especially in the activities of pectinolytic, cellulolytic and proteolytic enzymes and their capacity to macerate potato tuber tissue.     

Virulence of ‘Dickeya solani’ and Dickeya dianthicola biovar‐1 and ‐7 strains on potato (Solanum tuberosum)

Studies were conducted to explain the relative success of ‘Dickeya solani’, a genetic clade of Dickeya biovar 3 and a blackleg‐causing organism that, after recent introduction, has spread rapidly in seed potato production in Europe to the extent that it is now more frequently detected than D. dianthicola. In vitro experiments showed that both species were motile, had comparable siderophore production and pectinolytic activity, and that there was no antagonism between them when growing. Both ‘D. solani’ and biovar 1 and biovar 7 of D. dianthicola rotted tuber tissue when inoculated at a low density of 10³ CFU mL^?1. In an agar overlay assay, D. dianthicola was susceptible to 80% of saprophytic bacteria isolated from tuber extracts, whereas ‘D. solani’ was susceptible to only 31%, suggesting that ‘D. solani’ could be a stronger competitor in the potato ecosystem. In greenhouse experiments at high temperatures (28°C), roots were more rapidly colonized by ‘D. solani’ than by biovar 1 or 7 of D. dianthicola and at 30 days after inoculation higher densities of ‘D. solani’ were found in stolons and progeny tubers. In co‐inoculated plants, fluorescent protein (GFP or DsRed)‐tagged ‘D. solani’ outcompeted D. dianthicola in plants grown from vacuum‐infiltrated tubers. In 3 years of field studies in the Netherlands with D. dianthicola and ‘D. solani’, disease incidence varied greatly annually and with strain. In summary, ‘D. solani’ possesses features which allow more efficient plant colonization than D. dianthicola at high temperatures. In temperate climates, however, tuber infections with ‘D. solani’ will not necessarily result in a higher disease incidence than infections with D. dianthicola, but latent seed infection could be more prevalent.     

Virulence of Pectobacterium carotovorum subsp. brasiliense on potato compared with that of other Pectobacterium and Dickeya species under climatic conditions prevailing in the Netherlands

In western Europe, Pectobacterium carotovorum subsp. brasiliense is emerging as a causal agent of blackleg disease. In field experiments in the Netherlands, the virulence of this pathogen was compared with strains of other Dickeya and Pectobacterium species. In 2013 and 2014, seed potato tubers were vacuum infiltrated with high densities of bacteria (10⁶ CFU mL^?1) and planted in clay soil. Inoculation with P. carotovorum subsp. brasiliense and P. atrosepticum resulted in high disease incidences (75–95%), inoculation with D. solani and P. wasabiae led to incidences between 5% and 25%, but no significant disease development was observed in treatments with P. carotovorum subsp. carotovorum, D. dianthicola or the water control. Co‐inoculations of seed potatoes with P. carotovorum subsp. brasiliense and D. solani gave a similar disease incidence to inoculation with only P. carotovorum subsp. brasiliense. However, co‐inoculation of P. carotovorum subsp. brasiliense with P. wasabiae resulted in a decrease in disease incidence compared to inoculation with only P. carotovorum subsp. brasiliense. In 2015, seed potatoes were inoculated with increasing densities of P. carotovorum subsp. brasiliense, D. solani or P. atrosepticum (10³–10⁶ CFU mL^?1). After vacuum infiltration, even a low inoculum density resulted in high disease incidence. However, immersion without vacuum caused disease only at high bacterial densities. Specific TaqMan assays were evaluated and developed for detection of P. carotovorum subsp. brasiliense, P. wasabiae and P. atrosepticum and confirmed the presence of these pathogens in progeny tubers of plants derived from vacuum‐infiltrated seed tubers.     

Characterization of bacterial isolates from rotting potato tuber tissue showing antagonism to Dickeya sp. biovar 3 in vitro and in planta

Possibilities for biocontrol of biovar 3 Dickeya sp. in potato were investigated, using bacteria from rotting potato tissue isolated by dilution plating on nonselective agar media. In a plate assay, 649 isolates were screened for antibiosis against Dickeya sp. IPO2222 and for the production of siderophores. Forty‐one isolates (6·4%) produced antibiotics and 112 isolates (17·3%) produced siderophores. A selection of 41 antibiotic‐producing isolates and 41 siderophore‐producing isolates were tested in a potato slice assay for control of the Dickeya sp. Isolates able to reduce rotting of potato tuber tissue by at least 50% of the control were selected. Isolates were characterized by 16S rDNA analysis as Bacillus, Pseudomonas, Rhodococcus, Serratia, Obesumbacterium and Lysinibacillus genera. Twenty‐three isolates belonging to different species and genera, 13 producing antibiotics and 10 producing siderophores, were further characterized by testing acyl‐homoserine lactone (AHL) production, quorum quenching, motility, biosurfactant production, growth at low (4·0) and high (10·0) pH, growth at 10°C under aerobic and anaerobic conditions and auxin production. In replicated greenhouse experiments, four selected antagonists based on the in vitro tests were tested in planta using wounded or intact minitubers of cv. Kondor subsequently inoculated by vacuum infiltration with an antagonist and a GFP (green fluorescent protein)‐tagged biovar 3 Dickeya sp. strain. A potato endophyte A30, characterized as S. plymuthica, protected potato plants by reducing blackleg development by 100% and colonization of stems by Dickeya sp. by 97%. The potential use of S. plymuthica A30 for the biocontrol of Dickeya sp. is discussed.     

Pectinolytic bacteria associated with potato soft rot and blackleg in South Africa and Zimbabwe

Soft rot and blackleg can cause severe economic losses in potato production in South Africa and Zimbabwe depending on climatic conditions. The aim of the study was to identify the predominant bacteria causing potato soft rot and blackleg in these countries. Samples, comprising of stems and tubers from potato plants with blackleg and soft rot symptoms were collected from 2006?C2009 from potato production areas where disease outbreaks occurred. The isolates from these plants and tubers yielded Gram negative, pectinolytic bacteria on crystal violet pectate and inoculated tubers. Identification was based on biochemical and phenotypic characteristics, rep-PCR, Amplified Fragment Length Polymorphisms and sequences of gyrB and recA genes. Isolates from Zimbabwe were identified as Pectobacterium carotovorum subsp. brasiliensis (Pcb) (21 isolates), Dickeya dadantii subsp. dadantii (Dd) (20 isolates), P. c. subsp. carotovorum (Pcc) (16 isolates) and P. atrosepticum (Pa) (4 isolates). Pcb, Pcc and Dd subsp. dadantii were isolated from samples collected from all the regions, while Pa was isolated from Nyanga the coolest region in Zimbabwe. In South Africa, however, Pcb was the most common causal agent of soft rot and blackleg. P. atrosepticum was the only pathogen isolated from samples collected in Nyanga, Zimbabwe, and was not isolated from any South African samples. AFLP analysis separated the Pcb strains into 12 clusters, reflecting subdivision in terms of geographic origin, and Pcc isolates were clearly differentiated from Pcb isolates. A large degree of DNA polymorphism was evident among these 12 clusters. The study identified all the pathogens associated with the blackleg/soft rot disease complex.     

Studies on the interaction between the biocontrol agent,Serratia plymuthica A30, and blackleg‐causing Dickeya sp. (biovar 3) in potato (Solanum tuberosum)

Interactions between Serratia plymuthica A30 and a blackleg‐causing biovar 3 Dickeya sp. were examined. In a potato slice assay, S. plymuthica A30 inhibited tissue maceration caused by Dickeya sp. IPO2222 when co‐inoculated at a density at least 10 times greater than that of the pathogen. In glasshouse experiments, population dynamics of the antagonist and of the pathogen in planta were studied by dilution plating and confocal laser scanning microscopy (CLSM) using fluorescent protein‐tagged strains. Pathogen‐free minitubers were vacuum‐infiltrated with DsRed‐tagged Dickeya sp. IPO2222 and superficially treated during planting with a water suspension containing GFP‐tagged S. plymuthica A30. A30 reduced the blackleg incidence from 55% to 0%. Both the pathogen and the antagonist colonized the seed potato tubers internally within 1 day post‐inoculation (dpi). Between 1 and 7 dpi, the population of A30 in tubers increased from 10¹ to c. 10³ CFU g^?1 and subsequently remained stable until the end of the experiment (28 dpi). Populations of A30 in stems and roots increased from c. 10² to c. 10⁴ CFU g^?1 between 7 and 28 dpi. Dilution plating and CLSM studies showed that A30 decreased the density of Dickeya sp. populations in plants. Dilution plating combined with microscopy allowed the enumeration of strain A30 and its visualization in the vascular tissues of stem and roots and in the pith of roots, as well as its adherence to and colonization of the root surface. The implications of these finding for the use of S. plymuthica A30 as a biocontrol agent are discussed.     

Detection of phytopathogens of the genus Dickeya using a PCR primer prediction pipeline for draft bacterial genome sequences

This study used a novel computational pipeline to exploit draft bacterial genome sequences in order to predict, automatically and rapidly, PCR primer sets for Dickeya spp. that were unbiased in terms of diagnostic gene choice. This pipeline was applied to 16 draft and four complete Dickeya genome sequences to generate >700 primer sets predicted to discriminate between Dickeya at the species level. Predicted diagnostic primer sets for both D. dianthicola (DIA‐A and DIA‐B) and ‘D. solani’ (SOL‐C and SOL‐D) were validated against a panel of 70 Dickeya reference strains, representative of the known diversity of this genus, to confirm primer specificity. The classification of the four previously sequenced strains was re‐examined and evidence of possible misclassification of three of these strains is presented.     

Phylogenetic study of Japanese Dickeya spp. and development of new rapid identification methods using PCR–RFLP

Forty-one representative Japanese Dickeya spp. (Erwinia chrysanthemi) strains isolated from 24 plants in Japan were investigated using multilocus sequence analysis of recA, dnaX, rpoD, gyrB and 16S rDNA; PCR–RFLP (restriction fragment length polymorphism) of recA, rpoD and gyrB genes; PCR genomic fingerprinting; and biochemical tests. Based on the recA, dnaX, rpoD, gyrB and 16S rDNA sequences and PCR genomic fingerprinting, the strains were essentially divided into six groups (I–VI). Group I corresponded to D. chrysanthemi, group II corresponded to D. dadantii, group III to D. dianthicola and group IV to D. zeae. Meanwhile, group V and group VI could not be assigned to any existing Dickeya species, and they were deduced to be two putative new species. The PCR–RFLP analysis of gyrB, rpoD and recA clearly differentiated the six groups of Dickeya strains. From the results of the biochemical tests, the strains were assigned to biovars 1, 3, 5, 8 and 9; only one strain (SUPP 2525) was not assignable to the existing biovars. We also showed that the PCR–RFLP analysis of rpoD, gyrB and recA can be used as a rapid technique to identify Japanese Dickeya strains.