Characterization of Malaysian Pectobacterium spp. from vegetables using biochemical, molecular and phylogenetic methods

In August 2011, vegetable crops showing symptoms of maceration and water soaked lesions on their tuber, leaf, and fruit were collected from four major vegetable growing states in Malaysia including Pahang, Johor, Melaka and Selangor. The majority of the causal organisms isolated from infected tissues (52 strains) were identified as Pectobacterium spp. based on PCR amplification of the pectate lyase (pel) gene and amplification of the 16S-23S rRNA (ITS) with G1 and L1 primers. Physiological and biochemical assays divided Malaysian Pectobacterium species into two main groups: Pectobacterium wasabiae and Pectobacterium carotovorum subsp carotovorum. Partial sequence of PCR product from reaction of putative Pectobacterium spp. with 16S rRNA confirmed the results obtained from physiological and biochemical assays used for identification of the bacterium. Application of specific primers such as Eca1F/Eca2r, Br1f/L1r, EXPCCF/EXPCCR, and also ITS-PCR following by RFLP by restriction enzyme (RsaI) successfully differentiated Malaysian P. wasabiae and P. carotovorum subsp carotovorum isolates from other species and subspecies of Pectobacterium. Phylogenetic analysis of Malaysian isolates with housekeeping genes (mdh, gapA) grouped Malaysian P. carotovorum subsp carotovorum and P. wasabiae in the same cluster with P. carotovorum subsp carotovorum (Ecc380) and P. wasabiae (SCRI488) respectively.     

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.     

Occurrence of Pectobacterium wasabiae in potato field samples

During the growing seasons of 1996 and 1997, samples of potato stems and tubers with symptoms of blackleg and soft rot were collected in different regions in Poland. After growing to pure cultures on crystal violet pectate (CVP) medium, isolates of bacteria were identified as Pectobacterium spp. on the basis of their ability to degrade pectate and with the use of biochemical tests. About 43 % strains isolated from 122 different plant samples were identified as Pectobacterium carotovorum subsp. carotovorum, whereas the rest of the pectinolytic bacteria was identified as Pectobacterium atrosepticum. A recent screening of these isolates with recA PCR-RFLP allowed identification of 18 different RFLP groups within the tested P. c. subsp. carotovorum strains. The third largest group of the tested P. c. subsp. carotovorum strains (14 %), which were assigned to the profile 3 recA PCR-RFLP, was re-identified as Pectobacterium wasabiae (formerly Erwinia carotovora subsp. wasabiae) on the basis of recA and 16S rRNA genes sequences. About 50 % of P. wasabiae isolated from potato, in contrast to horseradish isolates of P. wasabiae, have an ability to grow at 37°C and some of them grow on media containing 5 % of NaCl. In a pathogenicity test with 11 strains of P. wasabiae these strains showed a high capacity to rot potato tubers.     

<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.     

Characterization of Pectobacterium carotovorum subsp. odoriferum causing soft rot of stored vegetables

Pectobacterium carotovorum subsp. odoriferum has been generally considered to have a narrow host range and has been isolated most often from chicory. Research was conducted to identify 91 Pectobacterium spp. strains isolated from different vegetables in Europe, North and South America, Asia, and Africa, and to compare their ability to cause disease in chicory and potato. Among the 91 strains, 22 strains from Europe were identified as P. c. subsp. odoriferum. Based on phylogenetic analysis of 16S rDNA, recA, and rpoS gene sequences, strains isolated from stored vegetables clustered together with the type strain of P. c. subsp. odoriferum and clustered separately from the P. c. subsp. carotovorum isolates. Eleven strains previously identified as P. c. subsp. carotovorum were reclassified as P. c. subsp. odoriferum. All P. c. subsp. odoriferum isolates were able to cause soft rot symptoms on chicory and potato. Moreover, the symptoms on potatoes were more severe at temperatures from 15 to 37 °C with P. c. subsp. odoriferum isolates than with P. atrosepticum or P. c. subsp. carotovorum isolates. Tissue maceration by P. c. subsp. odoriferum isolates was highest at 28 °C, and at that temperature tissue maceration was two-times greater for P. c. subsp. odoriferum isolates than for P. c. subsp. carotovorum isolates. Symptoms on inoculated chicory leaves were more severe with P. c. subsp. odoriferum (regardless of origin) than with other subspecies or species. To our knowledge, this is the first report that P. c. subsp. odoriferum occurs on a wide range of vegetables and has the ability to cause soft rot during potato storage.     

Genotypic and phenotypic variability of <Emphasis Type="Italic">Pectobacterium</Emphasis> strains causing blackleg and soft rot on potato in Turkey

Pectinolytic bacteria were isolated from 48 potato plants showing the symptoms of blackleg and collected in different fields of commercial potato production areas at Samsun, Amasya, Corum and Yozgat provinces in Turkey in 2015. The survey resulted in the isolation of 26 pectinolytic strains that belonged to P. atrosepticum, P. carotovorum subsp. brasiliense, P. carotovorum subsp. carotovorum and P. parmentieri species based on molecular identification with species-specific PCR and phenotypic characterization. The identified strains indicated typical biochemical characteristics of the assigned species. For 16 representative Pectobacterium isolates 10 unique rep-PCR band patterns were obtained. The 16S rRNA and recA and gapA gene fragment sequencing confirmed the species identity of the isolates. The phenotypic characterization of the strains revealed that for all assays but one (cellulase, protease activity, swimming but not swarming), the tested Pectobacterium species were significantly different from each other proving the diversity of the strains belonging to these genera. Recent outbreaks of blackleg and/or soft rot in potato production areas in Turkey may pose a threat on other crops, as tomato, pepper, cucumber, onion, cabbage, broccoli and sugar beet are cultivated in the same provinces.     

Role of co‐infection by Pectobacterium and Verticillium dahliae in the development of early dying and aerial stem rot of Russet Burbank potato

Potato early dying (PED) is a disease complex primarily caused by the fungus Verticillium dahliae. Pectolytic bacteria in the genus Pectobacterium can also cause PED symptoms as well as aerial stem rot (ASR) of potato. Both pathogens can be present in potato production settings, but it is not entirely clear if additive or synergistic interactions occur during co‐infection of potato. The objective of this study was to determine if co‐infection by V. dahliae and Pectobacterium results in greater PED or ASR severity using a greenhouse assay and quantitative real‐time PCR to quantify pathogen levels in planta. PED symptoms caused by Pectobacterium carotovorum subsp. carotovorum isolate Ec101 or V. dahliae isolate 653 alone included wilt, chlorosis and senescence and were nearly indistinguishable. Pectobacterium wasabiae isolate PwO405 caused ASR symptoms including water‐soaked lesions and necrosis. Greater Pectobacterium levels were detected in plants inoculated with PwO405 compared to Ec101, suggesting that ASR can result in high Pectobacterium populations in potato stems. Significant additive or synergistic effects were not observed following co‐inoculation with these strains of V. dahliae and Pectobacterium. However, infection coefficients of V. dahliae and Ec101 were higher and premature senescence was greater in plants co‐inoculated with both pathogens compared to either pathogen alone in both trials, and V. dahliae levels were greater in basal stems of plants co‐inoculated with either Pectobacterium isolate. Overall, these results indicate that although co‐infection by Pectobacterium and V. dahliae does not always result in significant additive or synergistic interactions in potato, co‐infection can increase PED severity.     

The use of two complementary DNA assays,AFLP and MLSA,for epidemic and phylogenetic studies of pectolytic enterobacterial strains with focus on the heterogeneous species Pectobacterium carotovorum

Amplified fragment length polymorphism (AFLP) markers and multilocus sequence analysis (MLSA) were used to analyse 63 bacterial strains, including 30 soft‐rot‐causing bacterial strains collected from Syrian potato fields and 33 reference strains. For the MLSA, additional sequences of 41 strains of Pectobacterium and Dickeya, available from the NCBI GenBank, were included to produce a single alignment of the 104 taxa for the seven concatenated genes (acnA, gapA, proA, icd, mtlD, mdh and pgi). The results indicate the need for a revision of the previously classified strains, as some potato‐derived Pectobacterium carotovorum strains were re‐identified as P. wasabiae. The strains that were classified as P. carotovorum during the analyses demonstrated high heterogeneity and grouped into five P. carotovorum highly supported clusters (PcI to PcV). The strains represented a wide range of host plants including potatoes, cabbage, avocados, arum lilies, sugar cane and more. Host specificity was detected in PcV, in which four of the six strains were isolated from monocotyledonous plants. The PcV strains formed a clearly distinct group in all the constructed phylogenetic trees. The number of strains phylogenetically classified as subspecies ‘P. c. subsp. brasiliensis’ in PcIV dramatically increased in size as a result of the characterization of new isolates or re‐identification of previous P. carotovorum and P. atrosepticum strains. The P. carotovorum strains from Syria were grouped into PcI, PcII and PcIV. This grouping indicates a lack of correlation between the geographical origin and classification of these pathogens.     

Characterization of Pectobacterium carotovorum subsp. carotovorum and brasiliense from diseased potatoes in Kenya

Using a DNA-based typing method, 48 bacterial strains isolated from infected potato (Solanum tuberosum) tubers originating from Kenya were characterized. The pel gene specific primers showed that all the 48 bacterial strains were pectolytic. Subspecies-specific primers EXPCCF/EXPCCR and Br1f/L1r identified 66 % of the strains as Pectobacterium carotovorum subsp. carotovorum while 34 % were identified as Pectobacterium carotovorum subsp. brasiliense based on their characteristic band sizes of 550 and 322 bp, respectively. Amplification of the 16S-23S rDNA (ITS) region did not yield observable differences in banding patterns between the Kenyan strains. However, PCR-RFLP analysis together with partial nucleotide sequences of the housekeeping mdh and gapA genes confirmed the results obtained by the specific primers. Phylogenetic analysis of the concatenated partial gene sequences grouped Pectobacterium carotovorum subsp. carotovorum and Pectobacterium carotovorum subsp. brasiliense Kenyan strains together with those identified in other parts of the world with 90 % and 99 % bootstrap support values, respectively. Pathogenicity assays using representative Kenyan strains demonstrated varied levels of tuber maceration ability. The Pectobacterium carotovorum subsp. carotovorum and Pectobacterium carotovorum subsp. brasiliense Kenyan strains were shown to be less aggressive in causing soft rot when compared to type strains. This study describes for the first time the genetic diversity of pectolytic bacteria causing soft rot disease of potatoes in Kenya.     

Pectobacterium spp. associated with bacterial stem rot syndrome of potato in Canada

Pectobacterium atrosepticum, P. carotovorum subsp. brasiliensis, P. carotovorum subsp. carotovorum, and P. wasabiae were detected in potato stems with blackleg symptoms using species- and subspecies-specific polymerase chain reaction (PCR). The tests included a new assay for P. wasabiae based on the phytase gene sequence. Identification of isolates from diseased stems by biochemical or physiological characterization, PCR, and multi-locus sequence typing (MLST) largely confirmed the PCR detection of Pectobacterium spp. in stem samples. P. atrosepticum was most commonly present but was the sole Pectobacterium sp. detected in only 52% of the diseased stems. P. wasabiae was most frequently present in combination with P. atrosepticum and was the sole Pectobacterium sp. detected in 13% of diseased stems. Pathogenicity of P. wasabiae on potato and its capacity to cause blackleg disease were demonstrated by stem inoculation and its isolation as the sole Pectobacterium sp. from field-grown diseased plants produced from inoculated seed tubers. Incidence of P. carotovorum subsp. brasiliensis was low in diseased stems, and the ability of Canadian strains to cause blackleg in plants grown from inoculated tubers was not confirmed. Canadian isolates of P. carotovorum subsp. brasiliensis differed from Brazilian isolates in diagnostic biochemical tests but conformed to the subspecies in PCR specificity and typing by MLST.     

Effect of a biofilm‐degrading enzyme from an oral pathogen in transgenic tobacco on the pathogenicity of Pectobacterium carotovorum subsp. carotovorum

The hypothesis that dispersin B (DspB), an enzyme from the periodontal pathogen Aggregatibacter actinomycetemcomitans that degrades the extracellular matrix polysaccharide PGA, will inhibit biofilm formation of the soft rot pathogen Pectobacterium carotovorum subsp. carotovorum in infected plants was tested by constitutive expression of DspB in tobacco plants. All the transgenic plants expressed properly folded and active DspB enzyme, although at different expression levels. In virulence assays, even the transgenic plant line D10, which produced a low level of DspB compared to other lines, showed significant resistance against P. carotovorum subsp. carotovorum, suggesting that DspB could be a valuable agent for biological control of P. carotovorum subsp. carotovorum infection in crop plants.     

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.     

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.     

<Emphasis Type="Italic">Pectobacterium carotovorum</Emphasis> subsp. <Emphasis Type="Italic">brasiliensis</Emphasis> causing blackleg on potatoes in South Africa

In South Africa during the 2006/2007 potato growing season, outbreaks of blackleg occurred, causing severe economic losses in commercial potato production fields. Symptoms were initially observed on only one stem per plant, on which the top leaves rolled upwards, wilted and became necrotic. As symptoms progressed to the lower leaves with subsequent leaf desiccation, a light to dark brown discolouration of the vascular system at the stem base developed, followed by external darkening. Under prevailing wet and humid conditions stems became slimy and pale. In the stems, the pith became necrotic and hollow. These symptoms were similar to those described in Brazil, where the causal agent was identified as a new subspecies, Pectobacterium carotovorum subsp. brasiliensis (Pbcb). Isolations from plants showing typical blackleg symptoms were made on CVP medium. Sequences and phylogenetic analysis of the partial 16S–23S rDNA intergenic spacer region indicated that the isolates were Pbcb. Comparison of PCR-RFLP patterns of the 16S–23S rDNA of isolates to reference cultures confirmed the identity of the South African blackleg strains as Pbcb, identical to strain 8 isolated in Brazil. This is the first report of Pbcb in South Africa and it appears to be the most important causal agent of blackleg in South Africa. The disease poses a major potential threat to the South African potato industry especially in terms of seed exports, tuber quality and yield.     

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.     

Evaluation of a diagnostic microarray for the detection of major bacterial pathogens of potato from tuber samples

Potato can be infected with many bacterial pathogens, the detection of which is necessary in seed certification. In this study, a diagnostic microarray previously tested for specificity of probes for detecting the potato bacteria causing blackleg and soft rot (Pectobacterium atrosepticum, Pectobacterium carotovorum, and Dickeya spp.), ring rot (Clavibacter. michiganensis subsp. sepedonicus), scab (Streptomyces scabies and Streptomyces turgidiscabies) and brown rot (Ralstonia solanacearum) from pure culture was evaluated for analytical sensitivity when testing directly from tuber samples. The microarray readily detected all the bacterial species when 100 ng of the target bacterial DNA from pure culture was mixed with DNA from soil microbes and potato. However, detection was inconsistent when total DNA isolated directly from infected tubers or enriched bacterial culture was used. While the high specificity of the probes could be confirmed from the results of the DNA cocktail experiment used as a control, the study demonstrated that the level of analytical sensitivity of the microarray under the tested condition was not sufficient to detect bacteria directly from tubers. Therefore, in addition to the cost and organizational complexities, the low analytical sensitivity and limited reproducibility of the microarray are constraints for establishing the platform for routine detection of potato bacterial pathogens from tuber samples.     

Characterization of <Emphasis Type="Italic">Pectobacterium carotovorum</Emphasis> subsp. <Emphasis Type="Italic">carotovorum</Emphasis> causing soft-rot disease on <Emphasis Type="Italic">Pinellia ternata</Emphasis> in China

Pinellia ternata is a traditional Chinese herb which has been used in China for over 1,000 years. A soft-rot disease characterized by water-soaked lesions and soft-rot symptoms with a stinking odour was commonly observed in cultivated fields of this plant, and Pectobacterium-like bacteria were consistently isolated from the infected tissues. Two typical strains (SXR1 and ZJR1), isolated from Shanxi and Zhejiang, respectively, were identified. Pathogenicity tests revealed that these strains were virulent to P. ternata and induced the same symptoms as observed in the field. Characterization involving fatty acid profile, metabolic and physiological properties, 16S rDNA sequence and PCR-RFLP identified both isolates as P. carotovorum subsp. carotovorum (Pcc). The 16S rDNA of both isolates shared 97–99% sequence similarity with that of Pcc strains. The phylogenetic trees showed that both isolates were clustered in the group of Pcc and P. carotovorum subsp. odorifera and both PCR-RFLP profiles were consistent with the pattern E produced by the minority of Pcc strains. Thus, isolates SXR1 and ZJR1 were characterized as Pcc in spite of some differences. This is the first report that Pcc has been proven as a causal agent of soft-rot disease on P. ternata.     

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.     

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.     

Characterization of Pectobacterium brasiliense strains from potato and vegetables in Israel

Pectobacterium brasiliense (Pbr) infects a wide range of crops worldwide, causing potato blackleg and soft rot and vegetable soft rots. This study aimed to characterize the genetic diversity and virulence variability among 68 Pbr strains isolated from either symptomless potato progeny tubers, diseased potato plants, ware potatoes wash water, or vegetables grown in Israel, as well as strains isolated from symptomless seed tubers grown in Europe, or diseased potato plants grown in France. The collection was typed using PCR and TaqMan real-time PCR analyses, dnaX sequence analysis, pulsed-field gel electrophoresis (PFGE), and pectolytic activity. dnaX phylogeny grouped almost all strains in a common genetic clade related to Pbr, which was distinct from the other Pectobacterium species. PFGE analysis identified two main clusters, including one major group of 47 strains with 95%–100% similarity. Maceration assays on two potato cultivars showed significant differences between strains but with no correlations with the source of the strains nor the status of the host (with/without symptoms). Molecular (dnaX sequences and PFGE profiles) and phenotypic analyses (tuber maceration tests) showed that the tested Pbr strains are not a homogeneous group. Analysis of the tested Pbr strains isolated from potato and vegetables grown in fields with a history of potato cultivation suggests that seed tubers imported from Europe may be the main source for Pbr in Israel. To the best of our knowledge, this is the first study that describes biodiversity and population structure of P. brasiliense isolated from potato and vegetables under hot climate conditions.