High genomic variability in the plant pathogenic bacterium Pectobacterium parmentieri deciphered from de novo assembled complete genomes.

BACKGROUND: Pectobacterium parmentieri is a newly established species within the plant pathogenic family Pectobacteriaceae. Bacteria belonging to this species are causative agents of diseases in economically important crops (e.g. potato) in a wide range of different environmental conditions, encountered in Europe, North America, Africa, and New Zealand. Severe disease symptoms result from the activity of P. parmentieri virulence factors, such as plant cell wall degrading enzymes. Interestingly, we observe significant phenotypic differences among P. parmentieri isolates regarding virulence factors production and the abilities to macerate plants. To establish the possible genomic basis of these differences, we sequenced 12 genomes of P. parmentieri strains (10 isolated in Poland, 2 in Belgium) with the combined use of Illumina and PacBio approaches. De novo genome assembly was performed with the use of SPAdes software, while annotation was conducted by NCBI Prokaryotic Genome Annotation Pipeline. RESULTS: The pan-genome study was performed on 15 genomes (12 de novo assembled and three reference strains: P. parmentieri CFBP 8475T, P. parmentieri SCC3193, P. parmentieri WPP163). The pan-genome includes 3706 core genes, a high number of accessory (1468) genes, and numerous unique (1847) genes. We identified the presence of well-known genes encoding virulence factors in the core genome fraction, but some of them were located in the dispensable genome. A significant fraction of horizontally transferred genes, virulence-related gene duplications, as well as different CRISPR arrays were found, which can explain the observed phenotypic differences. Finally, we found also, for the first time, the presence of a plasmid in one of the tested P. parmentieri strains isolated in Poland. CONCLUSIONS: We can hypothesize that a large number of the genes in the dispensable genome and significant genomic variation among P. parmentieri strains could be the basis of the potential wide host range and widespread diffusion of P. parmentieri. The obtained data on the structure and gene content of P. parmentieri strains enabled us to speculate on the importance of high genomic plasticity for P. parmentieri adaptation to different environments.

Draft Genome Sequence of a Highly Virulent Strain of the Plant Pathogen Dickeya solani, IFB0099.

Dickeya solani is an important bacterial pathogen of potato cultivars in Europe. Here, we present the draft genome of D. solani strain IFB0099 isolated from potato in Poland that shows a high level of pectinolytic activity and a high virulence. This genome sequence is 5,094,121 bp and contains 4,365 protein-coding sequences.

Detection, identification and differentiation of Pectobacterium and Dickeya species causing potato blackleg and tuber soft rot: a review.

The soft rot Enterobacteriaceae (SRE) Pectobacterium and Dickeya species (formerly classified as pectinolytic Erwinia spp.) cause important diseases on potato and other arable and horticultural crops. They may affect the growing potato plant causing blackleg and are responsible for tuber soft rot in storage thereby reducing yield and quality. Efficient and cost-effective detection and identification methods are essential to investigate the ecology and pathogenesis of the SRE as well as in seed certification programmes. The aim of this review was to collect all existing information on methods available for SRE detection. The review reports on the sampling and preparation of plant material for testing and on over thirty methods to detect, identify and differentiate the soft rot and blackleg causing bacteria to species and subspecies level. These include methods based on biochemical characters, serology, molecular techniques which rely on DNA sequence amplification as well as several less-investigated ones.

Simultaneous detection of major blackleg and soft rot bacterial pathogens in potato by multiplex polymerase chain reaction.

A multiplex polymerase chain reaction (PCR) assay for simultaneous, fast and reliable detection of the main soft rot and blackleg potato pathogens in Europe has been developed. It utilises three pairs of primers and enables detection of three groups of pectinolytic bacteria frequently found in potato, namely: Pectobacterium atrosepticum, Pectobacterium carotovorum subsp. carotovorum together with Pectobacterium wasabiae and Dickeya spp. in a multiplex PCR assay. In studies with axenic cultures of bacteria, the multiplex assay was specific as it gave positive results only with strains of the target species and negative results with 18 non-target species of bacteria that can possibly coexist with pectinolytic bacteria in a potato ecosystem. The developed assay could detect as little as 0.01 ng µL-1 of Dickeya sp. genomic DNA, and down to 0.1 ng µL-1 of P. atrosepticum and P. carotovorum subsp. carotovorum genomic DNA in vitro. In the presence of competitor genomic DNA, isolated from Pseudomonas fluorescens cells, the sensitivity of the multiplex PCR decreased tenfold for P. atrosepticum and Dickeya sp., while no change was observed for P. carotovorum subsp. carotovorum and P. wasabiae. In spiked potato haulm and tuber samples, the threshold level for target bacteria was 101 cfu mL-1 plant extract (102 cfu g-1 plant tissue), 102 cfu mL-1 plant extract (103 cfu g-1 plant tissue), 103 cfu mL-1 plant extract (104 cfu g-1 plant tissue), for Dickeya spp., P. atrosepticum and P. carotovorum subsp. carotovorum/P. wasabiae, respectively. Most of all, this assay allowed reliable detection and identification of soft rot and blackleg pathogens in naturally infected symptomatic and asymptomatic potato stem and progeny tuber samples collected from potato fields all over Poland.

Effective biotic elicitation of Ruta graveolens L. shoot cultures by lysates from Pectobacterium atrosepticum and Bacillus sp.

Growth of Ruta graveolens shoots was induced when Bacillus sp. cell lysates were added to the culture medium. Elicitation of coumarin by this lysate was also very effective; the concentrations of isopimpinelin, xanthotoxin and bergapten increased to 610, 2120 and 1460 microg g(-1) dry wt, respectively. It also had a significant effect on the production of psoralen and rutamarin (680 and 380 microg g(-1) dry wt) and induced the biosynthesis of chalepin, which was not detected in the control sample, up to 47 microg g(-1) dry wt With lysates of the Pectobacterium atrosepticum, their effect on growth was not so significant and had no effect on the induction of coumarin accumulation. But elicitation with this lysate was much more effective for inducing the production of furoquinolone alkaloids; the concentrations of gamma-fagarine, skimmianine, dictamnine and kokusaginine rose to 99, 680, 172 and 480 microg g(-1) dry wt, respectively.

Identification of a DNA restriction-modification system in Pectobacterium carotovorum strains isolated from Poland.

AIMS: Polish isolates of pectinolytic bacteria from the species Pectobacterium carotovorum were screened for the presence of a DNA restriction-modification (R-M) system. METHODS AND RESULTS: Eighty-nine strains of P. carotovorum were isolated from infected potato plants. Sixty-six strains belonged to P. carotovorum ssp. atrosepticum and 23 to P. carotovorum ssp. carotovorum. The presence of restriction enzyme Pca17AI, which is an isoschizomer of EcoRII endonuclease, was observed in all isolates of P. c. atrosepticum but not in P. c. carotovorum. The biochemical properties, PCR amplification, and sequences of the Pca17AI restriction endonuclease and methyltransferase genes were compared with the prototype EcoRII R-M system genes. Only when DNA isolated from cells of P. c. atrosepticum was used as a template, amplification of a 680 bp homologous to the gene coding EcoRII endonuclease. CONCLUSIONS: Endonuclease Pca17AI, having a relatively low temperature optimum, was identified. PCR amplification revealed that the nucleotide sequence of genes for EcoRII and Pca17AI R-M are different. Dcm methylation was observed in all strains of Pectobacterium and other Erwinia species tested. The sequence of a DNA fragment coding Dcm methylase in P. carotovorum was different from that of Escherichia coli. SIGNIFICANCE AND IMPACT OF THE STUDY: Pca17AI is the first psychrophilic isoschizomer of EcoRII endonuclease. The presence of specific Dcm methylation in chromosomal DNA isolated from P. carotovorum is described for the first time. A 680 bp PCR product, unique for P. c. atrosepticum strains, could serve as a molecular marker for detection of these bacteria in environmental samples.

Secondary metabolites in in vitro cultured plants of the genus Drosera.

Extracts from plantlets of different species of the genus Drosera, grown as in vitro cultures, were evaluated for the level of phenolic secondary metabolites from the group of naphthoquinones and flavonols. The profiles of natural products in the extracts obtained from different species were monitored by HPLC with UV detection at 260 and 330 nm. On the basis of the data obtained, Drosera binata, the species with the highest amount of plumbagin, was selected for further studies. The most effective method of extraction of quinones was established and the composition of phenolic secondary metabolites in the tissues was determined. For the identification of phenolic compounds, HPLC-UV and HPLC-ESI/MS were applied.

Stimulation of furanochromone accumulation in callus cultures of Ammi visnaga L. by addition of elicitors.

In order to check the possibility of producing secondary metabolites, in vitro cultures of A. visnaga callus were established. The best growth of A. visnaga callus was obtained on Murashige and Skoog medium (MS) containing 6-benzyladenine (BA) and alpha-naphtaleneacetic acid (NAA). The study was concentrated on the induction of production of secondary metabolites by exposing callus to abiotic elicitors: benzo(1,2,3)-thiadiazole-7-carbothionic acid S-methyl ester (BION) and a suspension of silica (SiO2) and biotic elicitors: autoclaved lysates of Enterobacter sakazaki and scleroglucan. GC analysis indicated that not-elicited callus of A. visnaga grown in darkness accumulated 2 times more visnagin than the one which was grown under a 16-h photoperiod. The highest accumulation of visnagin was observed in the callus culture elicited with scleroglucan or BION. Scleroglucan induced also the accumulation of khellin in A. visnaga callus. The presented work shows that biosynthesis of pharmacologically important secondary metabolites in A. visnaga cultures could be stimulated by application of elicitors.

Characterization of the pelL gene encoding a novel pectate lyase of Erwinia chrysanthemi 3937.

Erwinia chrysanthemi 3937 secretes five major isoenzymes of pectate lyases encoded by the pelA, pelB, pelC, pelD and pelE genes. Recently, a new set of pectate lyases was identified in E. chrysanthemi mutants deleted of those pel genes. We cloned the pelL gene, encoding one of these secondary pectate lyases of E. chrysanthemi 3937, from a genomic bank of a strain deleted of the five major pel genes. The nucleotide sequence of the region containing the pelL gene was determined. The pelL reading frame is 1275 bases long, corresponding to a protein of 425 amino acids including a typical amino-terminal signal sequence of 25 amino acids. Comparison of the amino acid sequences of PelL and the exo-pectate lyase PelX of E. chrysanthemi EC16 revealed a low homology, limited to 220 residues of the central part of the proteins. No homology was detected with other bacterial pectinolytic enzymes. Regulation of pelL transcription was analysed using gene fusion. As shown for the other pel genes, the transcription of pelL is dependent on various environmental conditions. It is induced by pectic catabolic products and affected by growth phase, temperature, iron starvation, osmolarity, anaerobiosis, nitrogen starvation and catabolite repression. Regulation of pelL expression appeared to be independent of the KdgR repressor, which controls all the steps of pectin catabolism. In contrast, the pecS gene, which is involved in regulation of the synthesis of the major pectate lyases and of cellulase, also appeared to be involved in pelL expression. The PelL protein is able to macerate plant tissue. This enzyme has a basic isoelectric point, presents an endo-cleaving activity on polygalacturonate or partially methylated pectin, with a basic pH optimum and an absolute requirement for Ca2+. The pelL mutant displayed a reduced virulence on potato tubers and Saintpaulia ionantha plants, demonstrating the important role of this enzyme in soft-rot disease.