Prophage-Derived Regions in Curtobacterium Genomes: Good Things, Small Packages.

Curtobacterium is a genus of Gram-positive bacteria within the order Actinomycetales. Some Curtobacterium species (C. flaccumfaciens, C. plantarum) are harmful pathogens of agricultural crops such as soybean, dry beans, peas, sugar beet and beetroot, which occur throughout the world. Bacteriophages (bacterial viruses) are considered to be potential curative agents to control the spread of harmful bacteria. Temperate bacteriophages integrate their genomes into bacterial chromosomes (prophages), sometimes substantially influencing bacterial lifestyle and pathogenicity. About 200 publicly available genomes of Curtobacterium species, including environmental metagenomic sequences, were inspected for the presence of sequences of possible prophage origin using bioinformatic methods. The comparison of the search results with several ubiquitous bacterial groups showed the relatively low level of the presence of prophage traces in Curtobacterium genomes. Genomic and phylogenetic analyses were undertaken for the evaluation of the evolutionary and taxonomic positioning of predicted prophages. The analyses indicated the relatedness of Curtobacterium prophage-derived sequences with temperate actinophages of siphoviral morphology. In most cases, the predicted prophages can represent novel phage taxa not described previously. One of the predicted temperate phages was induced from the Curtobacterium genome. Bioinformatic analysis of the modelled proteins encoded in prophage-derived regions led to the discovery of some 100 putative glycopolymer-degrading enzymes that contained enzymatic domains with predicted cell-wall- and cell-envelope-degrading activity; these included glycosidases and peptidases. These proteins can be considered for the experimental design of new antibacterials against Curtobacterium phytopathogens.

Curtobacterium spp. and Curtobacterium flaccumfaciens: Phylogeny, Genomics-Based Taxonomy, Pathogenicity, and Diagnostics.

The genus of Curtobacterium, belonging to the Microbacteriaceae family of the Actinomycetales order, includes economically significant pathogenic bacteria of soybeans and other agricultural crops. Thorough phylogenetic and full-genome analysis using the latest genomic data has demonstrated a complex and contradictory taxonomic picture within the group of organisms classified as the Curtobacterium species. Based on these data, it is possible to delineate about 50 new species and to reclassify a substantial part of the Curtobacterium strains. It is suggested that 53 strains, including most of the Curtobacterium flaccumfaciens pathovars, can compose a monophyletic group classified as C. flaccumfaciens. A genomic analysis using the most recent inventory of bacterial chromosomal and plasmid genomes deposited to GenBank confirmed the possible role of Microbacteriaceae plasmids in pathogenicity and demonstrated the existence of a group of related plasmids carrying virulence factors and possessing a gene distantly related to DNA polymerase found in bacteriophages and archaeal and eukaryotic viruses. A PCR diagnostic assay specific to the genus Curtobacterium was developed and tested. The presented results assist in the understanding of the evolutionary relations within the genus and can lay the foundation for further taxonomic updates.

Molecular, Phenotypical, and Host-Range Characterization of Robbsia andropogonis Strains Isolated from Bougainvillea spp. in Mexico.

Characteristic leaf spot and blight symptoms caused by Robbsia andropogonis on bougainvillea plants were found in three locations in different provinces of Mexico from 2019 to 2020. Eleven bacterial isolates with morphology similar to R. andropogonis were obtained from the diseased bougainvillea leaves. The isolates were confirmed as R. andropogonis by phenotypic tests and 16S rRNA, rpoD, and gyrB gene sequencing. In addition to bougainvillea, the strains were pathogenic to 10 agriculturally significant crops, including maize (Zea mays), sorghum (Sorghum bicolor), barley (Hordeum vulgare), coffee (Coffea arabiga), carnation (Dianthus caryophilus), Mexican lime (Citrus × aurantifolia), common bean (Phaseolus vulgaris), broadbeans (Vicia faba), and pea (Pisum sativum), but not runner bean (Phaseolus coccineus). The haplotypes network reveals the genetic variability among Mexican strains and its phylogeographic relationship with Japan, the U.S.A., and China. The presence of this pathogen represents a challenge for plant protection strategies in Mexico.

Ayka, a Novel Curtobacterium Bacteriophage, Provides Protection against Soybean Bacterial Wilt and Tan Spot.

Diseases caused by the Gram-positive bacterium Curtobacteriumflaccumfaciens pv. flaccumfaciens (Cff) inflict substantial economic losses in soybean cultivation. Use of specific bacterial viruses (bacteriophages) for treatment of seeds and plants to prevent the development of bacterial infections is a promising approach for bioprotection in agriculture. Phage control has been successfully tested for a number of staple crops. However, this approach has never been applied to treat bacterial diseases of legumes caused by Cff, and no specific bacteriophages have been known to date. This paper presents detailed characteristics of the first lytic bacteriophage infecting this pathogen. Phage Ayka, related to φ29-like (Salasmaviridae) viruses, but representing a new subfamily, was shown to control the development of bacterial wilt and tan spot in vitro and in greenhouse plants.

Pectobacterium versatile Bacteriophage Possum: A Complex Polysaccharide-Deacetylating Tail Fiber as a Tool for Host Recognition in Pectobacterial Schitoviridae.

Novel, closely related phages Possum and Horatius infect Pectobacterium versatile, a phytopathogen causing soft rot in potatoes and other essential plants. Their properties and genomic composition define them as N4-like bacteriophages of the genus Cbunavirus, a part of a recently formed family Schitoviridae. It is proposed that the adsorption apparatus of these phages consists of tail fibers connected to the virion through an adapter protein. Tail fibers possess an enzymatic domain. Phage Possum uses it to deacetylate O-polysaccharide on the surface of the host strain to provide viral attachment. Such an infection mechanism is supposed to be common for all Cbunavirus phages and this feature should be considered when designing cocktails for phage control of soft rot.

Autographivirinae Bacteriophage Arno 160 Infects Pectobacterium carotovorum via Depolymerization of the Bacterial O-Polysaccharide.

Phytopathogenic bacteria belonging to the Pectobacterium and Dickeya genera (soft-rot Pectobacteriaceae) are in the focus of agriculture-related microbiology because of their diversity, their substantial negative impact on the production of potatoes and vegetables, and the prospects of bacteriophage applications for disease control. Because of numerous amendments in the taxonomy of P. carotovorum, there are still a few studied sequenced strains among this species. The present work reports on the isolation and characterization of the phage infectious to the type strain of P. carotovorum. The phage Arno 160 is a lytic Podovirus representing a potential new genus of the subfamily Autographivirinae. It recognizes O-polysaccahride of the host strain and depolymerizes it in the process of infection using a rhamnosidase hydrolytic mechanism. Despite the narrow host range of this phage, it is suitable for phage control application.

Genomic characteristics of vB_PpaP_PP74, a T7-like Autographivirinae bacteriophage infecting a potato pathogen of the newly proposed species Pectobacterium parmentieri.

Bacteriophage vB_PpaP_PP74 (PP74) is a novel virulent phage that infects members of the species Pectobacterium parmentieri, a newly established species of soft-rot-causing bacteria in the family Pectobacteriaceae, derived from potato-specific Pectobacterium wasabiae. vB_PpaP_PP74 was identified as a member of the family Podoviridae by transmission electron microscopy. The phage has a 39,790-bp dsDNA genome containing 50 open reading frames (ORFs). Because of the absence of genes encoding toxins or lysogeny factors, PP74 may be considered a candidate phage for pathogen biocontrol applications. The genome layout is similar to genomes of T7-like phages within the subfamily Autographivirinae, and therefore, functions can be attributed to most of ORFs. However, the closest nucleotide sequence homologs of phage PP74 are unclassified Escherichia phages. Based on phylogenetic analysis, vB_PpaP_PP74 is a sensu lato T7-like phage, but it forms a distant subgenus group together with homologous enterobacterial phages.

Comprehensive analysis of draft genomes of two closely related pseudomonas syringae phylogroup 2b strains infecting mono- and dicotyledon host plants.

BACKGROUND: In recent years, the damage caused by bacterial pathogens to major crops has been increasing worldwide. Pseudomonas syringae is a widespread bacterial species that infects almost all major crops. Different P. syringae strains use a wide range of biochemical mechanisms, including phytotoxins and effectors of the type III and type IV secretion systems, which determine the specific nature of the pathogen virulence. RESULTS: Strains 1845 (isolated from dicots) and 2507 (isolated from monocots) were selected for sequencing because they specialize on different groups of plants. We compared virulence factors in these and other available genomes of phylogroup 2 to find genes responsible for the specialization of bacteria. We showed that strain 1845 belongs to the clonal group that has been infecting monocots in Russia and USA for a long time (at least 50 years). Strain 1845 has relatively recently changed its host plant to dicots. CONCLUSIONS: The results obtained by comparing the strain 1845 genome with the genomes of bacteria infecting monocots can help to identify the genes that define specific nature of the virulence of P. syringae strains.

Pb2MnTeO6 Double Perovskite: An Antipolar Anti-ferromagnet.

Pb2MnTeO6, a new double perovskite, was synthesized. Its crystal structure was determined by synchrotron X-ray and powder neutron diffraction. Pb2MnTeO6 is monoclinic (I2/m) at room temperature with a regular arrangement of all the cations in their polyhedra. However, when the temperature is lowered to ∼120 K it undergoes a phase transition from I2/m to C2/c structure. This transition is accompanied by a displacement of the Pb atoms from the center of their polyhedra due to the 6s(2) lone-pair electrons, together with a surprising off-centering of Mn(2+) (d(5)) magnetic cations. This strong first-order phase transition is also evidenced by specific heat, dielectric, Raman, and infrared spectroscopy measurements. The magnetic characterizations indicate an anti-ferromagnetic (AFM) order below TN ≈ 20 K; analysis of powder neutron diffraction data confirms the magnetic structure with propagation vector k = (0 1 0) and collinear AFM spins. The observed jump in dielectric permittivity near ∼150 K implies possible anti-ferroelectric behavior; however, the absence of switching suggests that Pb2MnTeO6 can only be antipolar. First-principle calculations confirmed that the crystal and magnetic structures determined are locally stable and that anti-ferroelectric switching is unlikely to be observed in Pb2MnTeO6.

Hole doping and structural transformation in CsTl1-xHgxCl3.

CsTlCl(3) and CsTlF(3) perovskites have been theoretically predicted to be superconductors when properly hole-doped. Both compounds have been previously prepared as pure compounds: CsTlCl(3) in a tetragonal (I4/m) and a cubic (Fm3̅m) perovskite polymorph and CsTlF(3) as a cubic perovskite (Fm3̅m). In this work, substitution of Tl in CsTlCl(3) with Hg is reported, in an attempt to hole-dope the system and induce superconductivity. The whole series CsTl(1-x)HgxCl(3) (x = 0.0, 0.1, 0.2, 0.4, 0.6, and 0.8) was prepared. CsTl(0.9)Hg(0.1)Cl(3) is tetragonal as the more stable phase of CsTlCl(3). However, CsTl(0.8)Hg(0.2)Cl(3) is already cubic with the space group Fm3̅m and with two different positions for Tl(+) and Tl(3+). For x = 0.4 and 0.5, solid solutions could not be formed. For x ≥ 0.6, the samples are primitive cubic perovskites with one crystallographic position for Tl(+), Tl(3+), and Hg(2+). All of the samples formed are insulating, and there is no signature of superconductivity. X-ray absorption spectroscopy indicates that all of the samples have a mixed-valence state of Tl(+) and Tl(3+). Raman spectroscopy shows the presence of the active Tl-Cl-Tl stretching mode over the whole series and the intensity of the Tl-Cl-Hg mode increases with increasing Hg content. First-principle calculations confirmed that the phases are insulators in their ground state and that Hg is not a good dopant in the search for superconductivity in this system.

Characterisation of New Foxunavirus Phage Murka with the Potential of Xanthomonas campestris pv. campestris Control.

Phages of phytopathogenic bacteria are considered to be promising agents for the biological control of bacterial diseases in plants. This paper reports on the isolation and characterisation of a new Xanthomonas campestris pv. campestris phage, Murka. Phage morphology and basic kinetic characteristics of the infection were determined, and a phylogenomic analysis was performed. The phage was able to lyse a reasonably broad range (64%, 9 of the 14 of the Xanthomonas campestris pv. campestris strains used in the study) of circulating strains of the cabbage black rot pathogen. This lytic myovirus has a DNA genome of 44,044 bp and contains 83 predicted genes. Taxonomically, it belongs to the genus Foxunavirus. This bacteriophage is promising for use as a possible means of biological control of cabbage black rot.

Phytopathogenic Curtobacterium flaccumfaciens Strains Circulating on Leguminous Plants, Alternative Hosts and Weeds in Russia.

Many bacterial plant pathogens have a broad host range important for their life cycle. Alternate hosts from plant families other than the main (primary) host support the survival and dissemination of the pathogen population even in absence of main host plants. Metabolic peculiarities of main and alternative host plants can affect genetic diversity within and between the pathogen populations isolated from those plants. Strains of Gram-positive bacterium Curtobacterium flaccumfaciens were identified as being causal agents of bacterial spot and wilt diseases on leguminous plants, and other crop and weed plants, collected in different regions of Russia. Their biochemical properties and susceptibility to copper compounds have been found to be relatively uniform. According to conventional PCR assays, all of the isolates studied were categorised as pathovar Curtobacterim flaccumfaciens pv. flaccumfaciens, a pathogen of legumes. However, the strains demonstrated a substantial diversity in terms of virulence on several tested host plants and different phylogenetic relationships were revealed by BOX-PCR and alanine synthase gene (alaS) sequencing.

Bacteriophage Control of Pseudomonas savastanoi pv. glycinea in Soybean.

Bacterial viruses (bacteriophages) have been considered as potential agents for the biological control of bacterial phytopathogens due to their safety and host specificity. Pseudomonas savastanoi pv. glycinea (Psg) is a causative agent of the bacterial spotting of soybean (Glycine max Willd). The harm caused by this bacterium to crop production and the development of antibiotic resistance in Psg and other pathogenic microorganisms has led to the pursuit of alternative management strategies. In this study, three Psg-specific lytic bacteriophages were isolated from soybean field soil in geographically distant regions of Russia, and their potential for protective action on plants was assessed. Sequencing of phage genomes has revealed their close relatedness and attribution to the genus Ghunavirus, subfamily Studiervirinae, family Autographiviridae. Extensive testing of the biological properties of P421, the representative of the isolated phage group, has demonstrated a relatively broad host range covering closely related Pseudomonas species and stability over wide temperature (4-40 °C) and pH (pH 4-7) ranges, as well as stability under ultraviolet irradiation for 30 min. Application of the phages to prevent, and treat, Psg infection of soybean plants confirms that they are promising as biocontrol agents.

Research of Water Molecules Cluster Structuring during Haberlea rhodopensis Friv. Hydration.

Gesneriaceae plant family is comprised of resurrection species, namely Boea hygrometrica and Paraboea rufescens, that are native to the Southeast Asia and Haberlea rhodopensis, Ramonda myconi, and Ramonda serbica, which are mainly found in the Balkan Peninsula. Haberlea rhodopensis is known to be able to survive extreme and prolonged dehydration. Study was carried out after the dried plant Haberlea rhodopensis Friv. had been hydrated and had reached its fresh state. Two juice samples were collected from the plant blossom: The first sample was prepared with 1% filtered water through a patented EVOdrop device. Then the sample was saturated with hydrogen with EVOdrop booster to a concentration of 1.2 ppm, pH = 7.3, ORP = -390 mV. This first sample was prepared with filtered tap water from Sofia, Bulgaria. The second sample, which was a control one, was developed with tap water from Sofia, Bulgaria, consisting of 1% solutions of Haberlea rhodopensis. A study revealed that during the drying process in H. rhodopensis the number of free water molecules decreases, and water dimers are formed. The aim of our study was to determine the number of water molecules in clusters in 1% solutions of hydrated H. rhodopensis plants. Results were analyzed according to the two types of water used in the experiment. Th EVOdrop device is equipped with an ultranano membrane and rotating jet nozzle to create a vortex water and saturation thanks to a second device EVObooster to obtain hydrogen-rich water. In the current study Hydrogen-rich water is referred to as Hydrogen EVOdrop Water (HEW). Research was conducted using the following methods-spectral methods non-equilibrium energy spectrum (NES) and differential non-equilibrium energy spectrum (DNES), mathematical models, and study of the distribution of water molecules in water clusters. In a licensed Eurotest Laboratory, the research of tap water before and after flowing through the EVOdrop device was proven. Studies have been carried out on the structuring of water molecule clusters after change of hydrogen bond energies. The restructuring comes with rearrangement of water molecules by the energy levels of hydrogen bonds. Local extrema can be observed in the spectrum with largest amount of water molecules. The structural changes were tested using the NES and DNES spectral methods. The conducted research proved that the application of EVOdrop device and EVObooster changes the parameters of water to benefit hydration and health.

Tailed Lytic Bacteriophages of Soft Rot Pectobacteriaceae.

The study of the ecological and evolutionary traits of Soft Rot Pectobacteriaceae (SRP) comprising genera Pectobacterium and Dickeya often involves bacterial viruses (bacteriophages). Bacteriophages are considered to be a prospective tool for the ecologically safe and highly specific protection of plants and harvests from bacterial diseases. Information concerning bacteriophages has been growing rapidly in recent years, and this has included new genomics-based principles of taxonomic distribution. In this review, we summarise the data on phages infecting Pectobacterium and Dickeya that are available in publications and genomic databases. The analysis highlights not only major genomic properties that assign phages to taxonomic families and genera, but also the features that make them potentially suitable for phage control applications. Specifically, there is a discussion of the molecular mechanisms of receptor recognition by the phages and problems concerning the evolution of phage-resistant mutants.

Molecular dynamics analysis of N-acetyl-D-glucosamine against specific SARS-CoV-2's pathogenicity factors.

The causative agent of the pandemic identified as SARS-CoV-2 leads to a severe respiratory illness similar to SARS and MERS with fever, cough, and shortness of breath symptoms and severe cases that can often be fatal. In our study, we report our findings based on molecular docking analysis which could be the new effective way for controlling the SARS-CoV-2 virus and additionally, another manipulative possibilities involving the mimicking of immune system as occurred during the bacterial cell recognition system. For this purpose, we performed molecular docking using computational biology techniques on several SARS-CoV-2 proteins that are responsible for its pathogenicity against N-acetyl-D-glucosamine. A similar molecular dynamics analysis has been carried out on both SARS-CoV-2 and anti-Staphylococcus aureus neutralizing antibodies to establish the potential of N-acetyl-D-glucosamine which likely induces the immune response against the virus. The results of molecular dynamic analysis have confirmed that SARS-CoV-2 spike receptor-binding domain (PDB: 6M0J), RNA-binding domain of nucleocapsid phosphoprotein (PDB: 6WKP), refusion SARS-CoV-2 S ectodomain trimer (PDB: 6X79), and main protease 3clpro at room temperature (PDB: 7JVZ) could bind with N-acetyl-D-glucosamine that these proteins play an important role in SARS-CoV-2's infection and evade the immune system. Moreover, our molecular docking analysis has supported a strong protein-ligand interaction of N-acetyl-D-glucosamine with these selected proteins. Furthermore, computational analysis against the D614G mutant of the virus has shown that N-acetyl-D-glucosamine affinity and its binding potential were not affected by the mutations occurring in the virus' receptor binding domain. The analysis on the affinity of N-acetyl-D-glucosamine towards human antibodies has shown that it could potentially bind to both SARS-CoV-2 proteins and antibodies based on our predictive modelling work. Our results confirmed that N-acetyl-D-glucosamine holds the potential to inhibit several SARS-CoV-2 proteins as well as induce an immune response against the virus in the host.

Quantitative Real-Time PCR Assay for the Detection of Pectobacterium parmentieri, a Causal Agent of Potato Soft Rot.

Pectobacterium parmentieri is a plant-pathogenic bacterium, recently attributed as a separate species, which infects potatoes, causing soft rot in tubers. The distribution of P. parmentieri seems to be global, although the bacterium tends to be accommodated to moderate climates. Fast and accurate detection systems for this pathogen are needed to study its biology and to identify latent infection in potatoes and other plant hosts. The current paper reports on the development of a specific and sensitive detection protocol based on a real-time PCR with a TaqMan probe for P. parmentieri, and its evaluation. In sensitivity assays, the detection threshold of this protocol was 102 cfu/mL on pure bacterial cultures and 102-103 cfu/mL on plant material. The specificity of the protocol was evaluated against P. parmentieri and more than 100 strains of potato-associated species of Pectobacterium and Dickeya. No cross-reaction with the non-target bacterial species, or loss of sensitivity, was observed. This specific and sensitive diagnostic tool may reveal a wider distribution and host range for P. parmentieri and will expand knowledge of the life cycle and environmental preferences of this pathogen.

Use of a Specific Phage Cocktail for Soft Rot Control on Ware Potatoes: A Case Study.

Using bacteriophages (bacterial viruses) to control pathogenic bacteria is a promising approach in horticulture. However, the application of this strategy in real conditions requires compliance with particular technological and environmental restraints. The presented paper concerns the process of phage selection to create a cocktail that is efficient against the circulating causal agents of potato soft rot. The resulting phage cocktail causes a complete lysis of a mixture of circulating pectobacterial strains in vitro. In the context of being used to treat ware potatoes during off-season storage, the protocol of phage application via the humidity maintenance system was designed. The phage cocktail was shown to reduce the population of Pectobacterium spp. 10-12-fold, achieving a population that was below a symptomatic threshold.

Development of qPCR Detection Assay for Potato Pathogen Pectobacterium atrosepticum Based on a Unique Target Sequence.

The recent taxonomic diversification of bacterial genera Pectobacterium and Dickeya, which cause soft rot in plants, focuses attention on the need for improvement of existing methods for the detection and differentiation of these phytopathogens. This research presents a whole genome-based approach to the selection of marker sequences unique to particular species of Pectobacterium. The quantitative real-time PCR assay developed is selective in the context of all tested Pectobacterium atrosepticum strains and is able to detect fewer than 102 copies of target DNA per reaction. The presence of plant DNA extract did not affect the sensitivity of the assay.