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.

Differentiation of myoblasts in culture: focus on serum and GABA.

There are many facts about the possible role of gamma-aminobutyric acid (GABA) in the development and differentiation of cells not only in nervous but also in muscle tissue. In the present study a primary culture of rat skeletal muscle myocytes was used to evaluate the correlation between the content of GABA in the cytoplasm and the processes of myocyte division and their fusion into myotubes.The effect of exogenous GABA on the processes of culture development was also estimated. Since the classical protocol for working with myocyte cultures involves the use of fetal bovine serum (FBS) to stimulate cell division (growth medium) and horse serum (HS) to activate the differentiation process (differentiation medium), the studies were carried out both in the medium with FBS and with HS. It was found that cells grown in medium supplemented withFBS contain more GABA compared to cultures growing in medium supplemented with HS. Addition of exogeneous GABA leads to a decrease in the number of myotubes formed in both media, while the addition of an amino acid to the medium supplemented with HS had a more pronounced inhibitory effect. Thus, we have obtained data indicating that GABA is able to participate in the early stages of skeletal muscle myogenesis by modulating the fusion process.

Occurrence, Characteristics, and qPCR-Based Identification of Pectobacterium versatile Causing Soft Rot of Chinese Cabbage in China.

Pectobacterium is one of the most important genera of phytopathogenic bacteria. It can cause soft-rot diseases on a wide range of plant species across the world. In this study, three Pectobacterium strains (KC01, KC02, and KC03) were isolated from soft-rotted Chinese cabbage in Beijing, China. These three strains were identified as Pectobacterium versatile based on phylogenetic analysis of Pectobacterium 16S ribosomal RNA, pmrA, and 504 Pectobacterium core genes, as well as a genomic average nucleotide identity analysis. Their biochemical characteristics were found to be similar to the P. versatile type strain ICMP9168T but differed in response to citric acid, stachyose, D-glucuronic acid, dextrin, and N-acetyl-ß-D-mannosamine. All of the tested P. versatile strains showed different carbohydrate utilization abilities compared with P. carotovorum and P. odoriferum, particularly in their ability to utilize D-arabitol, L-rhamnose, and L-serine. Under laboratory conditions, the maceration ability of P. versatile on Chinese cabbage was the highest at 28°C, compared with those at 13, 28, 23, and 33°C. Additionally, P. versatile could infect all of the 17 known Pectobacterium host plants, except for Welsh onion (Allium fistulosum). A SYBR Green quantitative PCR (qPCR) detection system was developed to distinguish P. versatile from other soft-rot bacteria based on the combined performance of melting curve (with a single melting peak at around 85°C) and fluorescence curve (with cycle threshold <30) when the bacterial genomic DNA concentration was in the range of 10 pg/µl to 10 ng/µl. This study is the first to report the presence of P. versatile on Chinese cabbage in China, as well as a specific and sensitive qPCR assay that can be used to quickly identify P. versatile. The work contributes to a better understanding of P. versatile and will facilitate the effective diagnosis of soft-rot disease, ultimately benefitting commercial crop production.

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.

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.

Soil Layers Impact Lithocarpus Soil Microbial Composition in the Ailao Mountains Subtropical Forest, Yunnan, China.

Plant litter decomposition is a complex, long-term process. The decomposition of litterfall is a major process influencing nutrient balance in forest soil. The soil microbiome is exceptionally diverse and is an essential regulator of litter decomposition. However, the microbiome composition and the interaction with litterfall and soil remain poorly understood. In this study, we examined the bacterial and fungal community composition of Lithocarpus across soil samples from different sampling seasons. Our results displayed that the microbiome assembly along the soil layer is influenced predominantly by the soil layer rather than by the sampling season. We identified that the soil layer strongly affected network complexity and that bacterial and fungal microbiomes displayed different patterns in different soil layers. Furthermore, source tracking and community composition analysis indicated that there are significantly different between soil and litter. Moreover, our results demonstrate that few dominant taxa (2% and 4% of bacterial and fungal phylotypes) dominated in the different soil layers. Hydnodontaceae was identified as the most important biomarker taxa for humic fragmented litter fungal microbiome and Nigrospora and Archaeorhizomycetaceae for organic soil and the organic mineral soil layer, and the phylum of Acidobacteria for the bacteria microbiome. Our work provides comprehensive evidence of significant microbiome differences between soil layers and has important implications for further studying soil microbiome ecosystem functions.

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.

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.

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.