Pectobacterium zantedeschiae sp. nov. a new species of a soft rot pathogen isolated from Calla lily (Zantedeschia spp.).

Four Gram-negative, rod-shaped pectinolytic bacterial strains designated as 2M, 9M, DPMP599 and DPMP600 were subjected to polyphasic analyses that revealed their distinctiveness from the other Pectobacterium species. Strains 2M and 9M were isolated from Calla lily bulbs cultivated in Central Poland. DPMP599 and DPMP600 strains were isolated from Calla lily leaves from plants grown in Serbia. Phylogenetic analyses based on nine housekeeping genes (gapA, gyrA, icdA, pgi, proA, recA, recN, rpoA, and rpoS), as well as phylogeny based on the 381 most conserved universal proteins confirmed that Pectobacterium zantedeschiae strains were distantly related to the other Pectobacterium, and indicated Pectobacterium atrosepticum, Pectobacterium betavasculorum, Pectobacterium parmentieri and Pectobacterium wasabiae as the closest relatives. Moreover, the analysis revealed that Pectobacterium zantedeschiae strains are not akin to Pectobacterium aroidearum strains, which were likewise isolated from Calla lily. The genome sequencing of the strains 2M, 9M and DPMP600 and their comparison with whole genome sequences of other Pectobacterium type strains confirmed their distinctiveness and separate species status within the genus based on parameters of in silico DNA-DNA hybridization and average nucleotide identity (ANI) values. The MALDI-TOF MS proteomic profile supported the proposition of delineation of the P. zantedeschiae and additionally confirmed the individuality of the studied strains. Based on of all of these data, it is proposed that the strains 2M, 9M, DPMP599, and DPMP600 isolated from Calla lily, previously assigned as P. atrosepticum should be reclassified as Pectobacterium zantedeschiae sp. nov. with the strain 9MT (PCM2893=DSM105717=IFB9009) as the type strain.

Characterization of Pectobacterium carotovorum proteins differentially expressed during infection of Zantedeschia elliotiana in vivo and in vitro which are essential for virulence.

The identification of phytopathogen proteins that are differentially expressed during the course of the establishment of an infection is important to better understand the infection process. In vitro approaches, using plant extracts added to culture medium, have been used to identify such proteins, but the biological relevance of these findings for in planta infection are often uncertain until confirmed by in vivo studies. Here, we compared the proteins of Pectobacterium carotovorum ssp. carotovorum strain PccS1 differentially expressed in Luria-Bertani medium supplemented with extracts of the ornamental plant Zantedeschia elliotiana cultivar 'Black Magic' (in vitro) and in plant tissues (in vivo) by two-dimensional electrophoresis coupled with mass spectrometry. A total of 53 differentially expressed proteins (>1.5-fold) were identified (up-regulated or down-regulated in vitro, in vivo or both). Proteins that exhibited increased expression in vivo but not in vitro, or in both conditions, were identified, and deletions were made in a number of genes encoding these proteins, four of which (clpP, mreB, flgK and eda) led to a loss of virulence on Z. elliotiana, although clpP and mreB were later also shown to be reduced in growth in rich and minimal media. Although clpP, flgK and mreB have previously been reported as playing a role in virulence in plants, this is the first report of such a role for eda, which encodes 2-keto-3-deoxy-6-phosphogluconate (KDPG) aldolase, a key enzyme in Entner-Doudoroff metabolism. The results highlight the value of undertaking in vivo as well as in vitro approaches for the identification of new bacterial virulence factors.

Cellvibrio zantedeschiae sp. nov., isolated from the roots of Zantedeschia aethiopica.

A bacterial strain, designated TPY-10T, was isolated from calla lily roots in Taiwan and characterized by using a polyphasic taxonomy approach. Cells of strain TPY-10T were Gram-stain-negative, strictly aerobic, motile and creamy white rods. Growth occurred at 15-35 °C (optimum, 25-30 °C), at pH 6-7 (optimum, pH 6) and with 0-1 % NaCl (optimum, 0 %). Phylogenetic analyses based on 16S rRNA gene sequences showed that strain TPY-10T belonged to the genus Cellvibrio and was most closely related to Cellvibriomixtus ACM 2601T with sequence similarity of 97.8 %. Strain TPY-10T contained C16 : 0, summed feature 3 (C16 : 1ω7c and/or C16 : 1ω6c) and C18 : 1ω7c as the predominant fatty acids. The only isoprenoid quinone was Q-9. The major polar lipids were phosphatidylethanolamine and phosphatidylglycerol. The DNA G+C content of the genomic DNA was 49.8 mol%. The DNA-DNA hybridization value for strain TPY-10T with Cellvibriomixtus ACM 2601T was less than 21 %. On the basis of the phylogenetic inference and phenotypic data, strain TPY-10T should be classified as a novel species, for which the name Cellvibrio zantedeschiae sp. nov. is proposed. The type strain is TPY-10T (=BCRC 80525T=LMG 27291T=KCTC 32239T).

Priming of protein expression in the defence response of Zantedeschia aethiopica to Pectobacterium carotovorum.

The defence response of Zantedeschia aethiopica, a natural rhizomatous host of the soft rot bacterium Pectobacterium carotovorum, was studied following the activation of common induced resistance pathways­systemic acquired resistance and induced systemic resistance. Proteomic tools were used, together with in vitro quantification and in situ localization of selected oxidizing enzymes. In total, 527 proteins were analysed by label-free mass spectrometry (MS) and annotated against the National Center for Biotechnology Information (NCBI) nonredundant (nr) protein database of rice (Oryza sativa). Of these, the fore most differentially expressed group comprised 215 proteins that were primed following application of methyl jasmonate (MJ) and subsequent infection with the pathogen. Sixty-five proteins were down-regulated following MJ treatments. The application of benzothiadiazole (BTH) increased the expression of 23 proteins; however, subsequent infection with the pathogen repressed their expression and did not induce priming. The sorting of primed proteins by Gene Ontology protein function category revealed that the primed proteins included nucleic acid-binding proteins, cofactor-binding proteins, ion-binding proteins, transferases, hydrolases and oxidoreductases. In line with the highlighted involvement of oxidoreductases in the defence response, we determined their activities, priming pattern and localization in planta. Increased activities were confined to the area surrounding the pathogen penetration site, associating these enzymes with the induced systemic resistance afforded by the jasmonic acid signalling pathway. The results presented here demonstrate the concerted priming of protein expression following MJ treatment, making it a prominent part of the defence response of Z. aethiopica to P. carotovorum.

Differential pathogenicity and genetic diversity among Pectobacterium carotovorum ssp. carotovorum isolates from monocot and dicot hosts support early genomic divergence within this taxon.

The capability of Pectobacterium carotovorum isolates to infect monocotyledonous plants has been previously reported; however, no full consideration was given to characterize the association between such isolates and their monocot hosts. To assess differences in aggressiveness among P. carotovorum ssp. carotovorum isolates originating from monocotyledonous or dicotyledonous plants, we used as model plants two susceptible monocot hosts, the ornamentals Zantedeschia aethiopica and Ornithogalum dubium, as well as two common dicot hosts, Solanum tuberosum and Brassica oleracea. Using virulence assays and different genetic analyses we characterized P. carotovorum ssp. carotovorum isolates from diverse geographical locations which originated from plants belonging to four unrelated orders of monocots and five orders of dicots. Invariably, isolates originating from monocots exhibited higher virulence towards the tested monocot plants than dicot isolates, independently of their geographical source. Moreover, monocot and dicot isolates were clearly differentiated by various genetic analyses, such as 16S rRNA sequence clustering, intergenic transcribed spacer-PCR (ITS-PCR) banding pattern and amplified fragment length polymorphism (AFLP). We propose that the observed relationship between pathogenicity and genetic diversity among P. carotovorum ssp. carotovorum isolates reveals a co-evolutionary specialization trend in the interaction between this pathogen and its hosts.

Priming of antimicrobial phenolics during induced resistance response towards Pectobacterium carotovorum in the ornamental monocot calla lily.

Calla lilies are herbaceous monocotyledonous plants that are highly sensitive to Pectobacterium carotovorum, the causal agent of soft-rot disease. Results demonstrate that, in response to elicitation using plant defense activators, the calla lily produces elevated levels of antimicrobial phenolics and that these compounds contribute to increased resistance against P. carotovorum, as shown by reduced bacterial proliferation in elicited leaves. The polyphenolic nature of the induced compounds was supported by autofluorescence, absorbance spectra, and reaction with Folin-Ciocalteu reagent. Two plant defense activators, Bion and methyl jasmonate, differed in both their capacity to induce accumulation of polyphenols and their resistance against the pathogen. Methyl jasmonate elicitation brought about higher accumulation of free phenolics relative to Bion, suggesting priming of bioactive polyphenols as a principal factor in the calla lily defense against P. carotovorum. To further characterize the nature of induced compounds, two major compounds were collected and identified as swertisin and isovitexin by mass and nuclear magnetic resonance spectroscopies.

Production of soft rot resistant calla lily by expressing a ferredoxin-like protein gene (pflp) in transgenic plants.

An efficient protocol for the Agrobacterium tumefaciens-mediated transformation of calla lily (Zantedeschia elliottiana (W. Wats.) Engl. cultivar 'Florex Gold') is described. Shoot basal discs were co-cultivated with A. tumefaciens C58C1 carrying a plasmid containing neomycin phosphotransferase (nptII) and plant ferredoxin-like protein (pflp) genes. After Agrobacterium co-cultivation, the shoot basal discs were exposed to 100 mg l(-1) kanamycin for selection. Twenty-eight out of 260 discs (10.8%) were found to have survived and produced shoot clusters. Twenty-six of these were confirmed to contain the pflp transgene by PCR, ending up in 10% transformation efficiency. The disease resistance investigation revealed that 18 transgenic plants exhibited resistance to soft rot disease caused by Erwinia carotovora subsp. carotovora. The presence of pflp gene was demonstrated by PCR, and its accumulation and activity was confirmed by Western blot and disease resistance assay. This was the first report to show the successful transformation and resistance to a bacterial pathogen in Zantedeschia. The protocol is useful for the quality improvement of calla lily through genetic transformation.