Agent Of Soft Rot Disease Research Articles

DIVERSITY OF BACTERIOPHAGES INFECTING PECTOBACTERIUM FROM POTATO FIELDS

Bacteria belonging to the genus Pectobacterium are causal agents of soft rot disease in diverse crops including potato. This disease is very difficult to control and no promising methods have been developed. The use of phage therapy has been suggested to control bacterial diseases; however, this method has not been applied widely because of its unreliability to the plant environment and the narrow spectrum of activity intrinsic to phages. This study identified bacteriophages capable of lysing diverse Pectobacterium species and isolates from plant and soil samples obtained from the potato rhizosphere. Repeated pickings of plaque resulted in the isolation of 189 single phages showing a diverse spectrum of host specificity. From these, 24 core phages were selected and a dendrogram was generated. A phylogenetic tree was also generated with the DNA fingerprints confirming the genetic diversity of the selected phages. The results of the phylogenetic tree almost fit those of the dendrogram. The stability of the selected phages was investigated under various temperatures and pH conditions in vitro. The phages were stable at 16°C-40°C and pH 6-7. Moreover, the viability of the phages differed significantly depending on the phage samples selected. Morphological characterization of the selected phages showed that they could be classified into three different groups, Podoviridae, Myoviridae, and Siphoviridae with diverse head sizes and tail lengths. These results are expected to contribute to controlling the disease, but further investigations of the genetic and molecular aspects will facilitate a better understanding of phage and bacteria interaction.

Plant phenolic volatiles inhibit quorum sensing in pectobacteria and reduce their virulence by potential binding to ExpI and ExpR proteins.

Quorum sensing (QS) is a population density-dependent regulatory system in bacteria that couples gene expression to cell density through accumulation of diffusible signaling molecules. Pectobacteria are causal agents of soft rot disease in a range of economically important crops. They rely on QS to coordinate their main virulence factor, production of plant cell wall degrading enzymes (PCWDEs). Plants have evolved an array of antimicrobial compounds to anticipate and cope with pathogens, of which essential oils (EOs) are widely recognized. Here, volatile EOs, carvacrol and eugenol, were shown to specifically interfere with QS, the master regulator of virulence in pectobacteria, resulting in strong inhibition of QS genes, biofilm formation and PCWDEs, thereby leading to impaired infection. Accumulation of the signal molecule N-acylhomoserine lactone declined upon treatment with EOs, suggesting direct interaction of EOs with either homoserine lactone synthase (ExpI) or with the regulatory protein (ExpR). Homology models of both proteins were constructed and docking simulations were performed to test the above hypotheses. The resulting binding modes and docking scores of carvacrol and eugenol support potential binding to ExpI/ExpR, with stronger interactions than previously known inhibitors of both proteins. The results demonstrate the potential involvement of phytochemicals in the control of Pectobacterium.

Efficacies of quorum sensing inhibitors, piericidin A and glucopiericidin A, produced by Streptomyces xanthocidicus KPP01532 for the control of potato soft rot caused by Erwinia carotovora subsp. atroseptica

To discover potential inhibitors of the quorum sensing (QS) system, a library of microbial culture extracts was screened with Chromobacterium violaceumCV026 strain. The culture extract of Streptomyces xanthocidicus KPP01532 contained quorum-sensing inhibitors (QSIs) of the CV026 strain. The active constituents of the culture extract of strain KPP01532 were purified using a series of chromatographic procedures, and based on data from NMR and mass spectroscopy, piericidin A and glucopiericidin A were identified. Erwinia carotovora subsp. atroseptica (Eca) is a plant pathogen that causes blackleg and soft rot diseases on potato stems and tubers. The virulence factors of Eca are regulated by QS. The expression of virulence genes (pelC, pehA, celV and nip) under the control of QS was monitored using quantitative real-time PCR (qRT-PCR). The transcription levels of the four genes were significantly lower when Eca was exposed to piericidin A or glucopiericidin A. These two compounds displayed similar control efficacies against soft rot caused by Eca in potato slices as furanone C-30. Therefore, piericidin A and glucopiericidin A are potential QSIs that suppress the expression of the virulence genes of Eca, suggesting that they could have potential use as control agents of soft rot disease on potato tubers.

Characterization of Pectobacterium carotovorum subsp. carotovorum Causing Watermelon Soft Rot Disease in Iran

AbstractThe aim of this study was characterized Pectobacterium carotovorum subsp. carotovorum (Pcc) the causal pathogen of watermelon soft rot disease in Iran. Of fifty bacterial isolates with white grey and convex colonies on nutrient agar obtained from symptomatic watermelon, ten isolates were selected for further tests. Pathogenicity tests results showed that all test isolates developed typical water‐soak symptoms after 2 days and signs of soft rot began 4 days after inoculation on watermelon fruits. Based on the phenotypic properties, the isolates were identified as Pectobacterium carotovorum subsp. carotovorum. The 16S rDNA sequences of isolates were 99% similar to the corresponding 16S rDNA sequence of the reference Pcc isolate. BOX and ERIC‐PCR analysis indicated that genetic diversity was present among the isolated Pcc isolates did not relate to the geographic location isolated from. To the best of our knowledge, this is the first study of biochemical and genotypic characterization of Pcc isolates the causal agents of soft rot disease on watermelon, in Iran.

Characterization of major hydrolytic enzymes secreted by Pythium myriotylum, causative agent for soft rot disease.

Pythium myriotylum, an oomycetous necrotroph is the causal agent of soft rot disease affecting several crops. Successful colonization by necrotrophs depends on their secretion of a diverse array of plant cell wall degrading enzymes (CWDEs). The induction dynamics of CWDEs secreted by P. myriotylum was analysed as little information is available for this pathogen. Activities of CWDEs that included pectinase, cellulase, xylanase and protease were detected using radial diffusion assay and differential staining. In Czapek Dox minimal medium supplemented with respective substrates as carbon source, the increase in CWDE activities was observed till 8days of incubation after which a gradual decline in enzymatic activities was observed. With sucrose as sole carbon source, all the enzymes studied showed increase in activity with fungal growth while with cell wall material derived from ginger rhizome as sole carbon source, an initial spurt in cellulase, xylanase and pectinase activities was observed 3days post incubation while protease activity increased from three days of incubation and reached maximum at 13days of incubation. To further evaluate the role of CWDEs in pathogenicity, UV-induced mutants (pmN14uv1) were generated wherein significant reduction in cellulase, pectinase and protease activities were observed while that of xylanase remained unchanged compared to wild type isolate (RGCBN14). Bioassays indicated changes in infection potential of pmN14uv1 thereby suggesting the crucial role played by P. myriotylum CWDEs in initiating the rotting process. Hence appropriate strategies that target the production/activity of these secretory hydrolytic enzymes will help in reducing disease incidence/pathogen virulence.

The influence of carbon source and calcium on the production of proteases by Erwinia chrysanthemi

Erwinia chrysanthemi (Ec) is the causative agent of soft rot diseases in many plants. In order to cause disease, it produces hydrolytic enzymes, which are involved in the degradation of plant tissue. However, the role of proteases in its pathogenicity to plants is not well understood. E. chrysanthemi targets the host’s middle lamella, which was mainly made of pectic substances containing calcium, for colonization. In this study, it is reported that a pectic substance, such as sodium polypectate (NaPP) may be preferable, not only for the production of hydrolytic enzymes, but for the production of protease as well. Furthermore, since the middle lamella contains high levels of calcium, it was thought that it may play an important role in the production of proteases by this organism. Both predictions were supported by the results presented in this study. When E. chrysanthemi subsp. chrysanthemi (Ecc) was grown in HXC medium supplemented with NaPP and calcium, it produced the highest levels of protease as compared to NaPP alone. These results suggest that proteases produced by E. chrysanthemi may be induced in part by calcium. Key words: Erwinia chrysanthem i, protease, phytopathogenicity, extracellular enzyme.

The Tat pathway of plant pathogen Dickeya dadantii 3937 contributes to virulence and fitness.

Protein secretion plays a very important role in the virulence of the bacterium Dickeya dadantii, the causative agent of soft rot disease, in a wide range of plant species. We studied the contribution of the twin-arginine translocation (Tat) protein system to the adaptation of D. dadantii 3937 to different growth conditions and to the interaction with the plant host. First, a list of 44 putative Tat substrates was obtained using bioinformatic programs taking advantage of the availability of the complete sequence of this bacterium. Second, a tatC mutant strain was constructed and analysed. The mutant displayed a pleiotropic phenotype, showing limited growth in an iron-depleted medium, higher sensitivity to copper, reduced motility on soft agar plates and attenuated virulence in witloof chicory leaves. Our results indicate the Tat system as an important determinant of the virulence and fitness of D. dadantii 3937. Potential Tat substrates related to the tatC mutant phenotype are discussed.

Expression of a Chimeric Magainin Gene in Potato Confers Improved Resistance to the Phytopathogen Erwinia carotovora

Magainin peptides originally identified from Xenopus laevis have cytotoxic effects against a range of prokary- otic organisms without harmful effects on higher eukaryotes. The mechanism of cytotoxicity of the peptides is by disrup- tion of membranes, which causes osmolysis. Magainin peptides are known to inhibit the in vitro growth of many phytopa- thogens including Erwinia carotovora, the causative agent of soft rot disease in potato. A synthetic gene was constructed based on the antimicrobial peptide magainin II. The coding sequence for the modified magainin II, magaininD, was con- structed using potato codon preference. Modifications included a point mutation previously shown to reduce proteolytic cleavage, and three substitutions known to increase activity of the peptide against prokaryotic organisms. Agrobacterium- mediated transformation was used to generate transgenic potato plants. Each line was tested for expression of the trans- gene at RNA and protein levels using RT-PCR and western analyses. Lines were identified that expressed both RNA and protein of the magaininD transgene. Field trials with the transgenic magaininD potato plants were conducted over three seasons, and harvested tubers were evaluated in bioassays for resistance to Erwinia carotovora ssp. atroseptica. Results from bioassays identified potato lines with significantly improved resistance to soft rot compared with control lines. The same lines were determined to have higher levels of expression of the transgene-derived peptide. The result demonstrated that the design, construction and transformation of a synthetic antimicrobial magainin gene was a successful strategy in introducing a novel form of disease resistance in potato plants.

The Crystal Structure of Pectate Lyase PelI from Soft Rot Pathogen Erwinia chrysanthemi in Complex with Its Substrate

The crystallographic structure of the family 3 polysaccharide lyase (PL-3) PelI from Erwinia chrysanthemi has been solved to 1.45 A resolution. It consists of an N-terminal domain harboring a fibronectin type III fold linked to a catalytic domain displaying a parallel beta-helix topology. The N-terminal domain is located away from the active site and is not involved in the catalytic process. After secretion in planta, the two domains are separated by E. chrysanthemi proteases. This event turns on the hypersensitive response of the host. The structure of the single catalytic domain determined to 2.1 A resolution shows that the domain separation unveils a "Velcro"-like motif of asparagines, which might be recognized by a plant receptor. The structure of PelI in complex with its substrate, a tetragalacturonate, has been solved to 2.3 A resolution. The sugar binds from subsites -2 to +2 in one monomer of the asymmetric unit, although it lies on subsites -1 to +3 in the other. These two "Michaelis complexes" have never been observed simultaneously before and are consistent with the dual mode of bond cleavage in this substrate. The bound sugar adopts a mixed 2(1) and 3(1) helical conformation similar to that reported in inactive mutants from families PL-1 and PL-10. However, our study suggests that the catalytic base in PelI is not a conventional arginine but a lysine as proposed in family PL-9.

The GacA global regulator is required for the appropriate expression of Erwinia chrysanthemi 3937 pathogenicity genes during plant infection

Pathogenicity of the phytopathogenic enterobacterium Erwinia chrysanthemi, the causal agent of soft rot disease on many plants, is a complex process involving several factors whose production is regulated by a complex, intertwined regulatory network. In this work we characterized the GacA regulator, member of the GacS-GacA two-component system, as a global regulator which is required for disease expression but not for bacterial multiplication in planta during the first stages of the plant infection. GacA was shown to control the expression of plant cell wall-degrading enzymes and hrp genes in vitro. Analysis of virulence gene expression during infection of Arabidopsis thaliana revealed a coordinated expression of these virulence genes at 12 h post infection and showed that GacA is required for the appropriate production of virulence factors in planta. GacA might partly act by negatively controlling the expression of the pecT gene encoding the global repressor PecT, indicating a hierarchy in the pathways involved in the E. chrysanthemi regulatory network.

Erwinia carotovora pv. carotovora, agente causante de la podredumbre blanda en algunas cosechas en Puerto Rico

Erwinia carotovora pv. carotovora es el causante de la podredumbre de cultivares en Puerto Rico. Se estudió el organismo causante de la podredumbre blanda aislado en tabaco, tomate, yautía, lechuga repollada, repollo y las ornamentales Pothos sp., Dracaena sp. y lirio cala. Las características fisiológicas, reacciones bioquímicas y sensitividad a antibióticos fueron similares entre los distintos aislamientos obtenidos. Pruebas de patogenicidad demostraron síntomas de podredumbre blanda en las distintas hospederas inoculadas. El organismo se identificó como E. carotovora pv. carotovora (Jones).