Plasmids are so closely associated with pathogens and antibiotic resistance that their potential for conferring other traits is often overlooked. Few studies consider how the full suite of traits encoded by plasmids is related to a host\'s environmental adaptation, particularly for Gram-positive bacteria. To investigate the role that plasmid traits might play in microbial communities from natural ecosystems, we identified plasmids carried by isolates of Curtobacterium (phylum Actinomycetota) from a variety of soil environments. We found that plasmids were common, but not ubiquitous, in the genus and varied greatly in their size and genetic diversity. There was little evidence of phylogenetic conservation among Curtobacterium plasmids even for closely related bacterial strains within the same ecotype, indicating that horizontal transmission of plasmids is common. The plasmids carried a wide diversity of traits that were not a random subset of the host chromosome. Furthermore, the composition of these plasmid traits was associated with the environmental context of the host bacterium. Together, the results indicate that plasmids contribute substantially to the microdiversity of a soil bacterium and that this diversity may play a role in niche differentiation and a bacterium\'s adaptation to its local environment.

Microbial communities are not the easiest to manipulate experimentally in natural ecosystems. However, leaf litter-topmost layer of surface soil-is uniquely suitable to investigate the complexities of community assembly. Here, we reflect on over a decade of collaborative work to address this topic using leaf litter as a model system in Southern California ecosystems. By leveraging a number of methodological advantages of the system, we have worked to demonstrate how four processes-selection, dispersal, drift, and diversification-contribute to bacterial and fungal community assembly and ultimately impact community functioning. Although many dimensions remain to be investigated, our initial results demonstrate that both ecological and evolutionary processes occur simultaneously to influence microbial community assembly. We propose that the development of additional and experimentally tractable microbial systems will be enormously valuable to test the role of eco-evolutionary processes in natural settings and their implications in the face of rapid global change.

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.

Soybeans are a valuable food product, containing 40% protein and a large percentage of unsaturated fatty acids ranging from 17 to 23%. Pseudomonas savastanoi pv. glycinea (Psg) and Curtobacterium flaccumfaciens pv. flaccumfaciens (Cff) are harmful bacterial pathogens of soybean. The bacterial resistance of soybean pathogens to existing pesticides and environmental concerns requires new approaches to control bacterial diseases. Chitosan is a biodegradable, biocompatible and low-toxicity biopolymer with antimicrobial activity that is promising for use in agriculture. In this work, a chitosan hydrolysate and its nanoparticles with copper were obtained and characterized. The antimicrobial activity of the samples against Psg and Cff was studied using the agar diffusion method, and the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were determined. The samples of chitosan and copper-loaded chitosan nanoparticles (Cu2+ChiNPs) significantly inhibited bacterial growth and were not phytotoxic at the concentrations of the MIC and MBC values. The protective properties of chitosan hydrolysate and copper-loaded chitosan nanoparticles against soybean bacterial diseases were tested on plants in an artificial infection. It was demonstrated that the Cu2+ChiNPs were the most effective against Psg and Cff. Treatment of pre-infected leaves and seeds demonstrated that the biological efficiencies of (Cu2+ChiNPs) were 71% and 51% for Psg and Cff, respectively. Copper-loaded chitosan nanoparticles are promising as an alternative treatment for bacterial blight and bacterial tan spot and wilt in soybean.

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 flaccumfaciens pv. flaccumfaciens (H.) Collins & Jones is known as a pathogen of different legume crops, including soybean (Glycine max (L.) Merr.) (Hedges 1922; Dunleavy 1983). OEPP/EPPO (2011) considers C. flaccumfaciens pv. flaccumfaciens as present in Russia based on reports of the disease on common beans in two regions of Russia (North Caucasus and Far East) made without proper pathogen identification. During the summer of 2020 and the spring of 2021, soybean plants with tan spot disease (10-40% of plants) were reported during routine assays of several fields in Stavropol Krai (44.72°N, 43.29°E). After harvest in 2021, we inspected 48 soybean seed lots collected in different regions of Russia for the presence of C. flaccumfaciens pv. flaccumfaciens. Seed testing was performed using the OEPP/EPPO (2011) protocol. For bacteria isolation, seed extracts were spread on MSCFF agar plates (Maringoni et al. 2006). After 5 days of incubation at 28°C potential, C. flaccumfaciens pv. flaccumfaciens colonies were used for further tests on NSA and SSM agar (Tegli et al. 2017, Maringoni et al. 2016). Six seed lots produced in Stavropol, Ryazan (53.95°N, 40.62°E), Orel (52.39°N, 37.69°E) and Amur (51.31°N, 128.22°E) regions were suspect. Ten isolates (SB1 to SB4 from Stavropol, F-125-1 to F-125-3 from Ryazan, and F-30-1 to F-30-3 from Amur) were selected, and further identified by morphological, physiological, and biochemical properties, MALDI TOF MS, 16S rRNA sequences, and specific primers CffFOR2 and CffREV4 (Tegli et al. 2017). Isolates consistently formed yellow, circular, smooth colonies on agar, and were identical to C. flaccumfaciens pv. flaccumfaciens type strain DSM 20129T in diagnostic physiological properties (Tegli et al. 2017). DNA was isolated from the bacteria by the CytoSorb Kit (Sintol, Moscow). All tested strains were positive in the PCR assay (Fig. 1). 16S rRNA fragments were amplified using primers 27F/1492R (Marchesi et al. 1998) and PCR products were sequenced (Evrogen, Moscow, Russia). The obtained 16S rRNA sequences (1473 bp, Accession No. OL539808.1-OL539817.1) were 100% identical to DSM 20129T (AM410688.1) according to a BLAST NCBI search. A pathogenicity test was done by leaf-cutting with scissors wetted with inoculum (for soybeans) or by injecting 5 microliters of the bacterial suspension (108 CFU/ml) into the stem (for common beans). All ten isolates for the inoculum were grown on nutrient agar for 72 h at 28°C. Soybean cv. Kasatka plants (stage V1) were used for inoculation, and common bean (cv. Purpurnaya) plants were inoculated as well to confirm multi-host virulence. Sterile water served as a control. Ten plantlets were used as replicates for each treatment. The plants were incubated at 24°C, 80% RH, and a 14 hour light/10 hour dark cycle. Tan spots (soybean) and wilt (beans) have developed 7-21 d.p.i (Fig. 2.1-2.6). Control plants remained asymptomatic. Seed inoculation by soaking them in the same bacterial suspension repeatedly produced twisted primary root (Fig. 2.7-2.8), but typical disease symptoms on leaves developed in 4-5 weeks only. The pathogen was successfully reisolated from all infected plants and not from the controls, thus fulfilling Koch\'s postulates. The identity of the reisolated strains was confirmed using morphological and physiological characteristics and the DNA sequence data for the 16S rRNA. These results indicated that a causal agent of the tan spot is present on soybean in three important agricultural areas of Russia (South, Central, and the Far East). To the best of our knowledge, this is the first report of C. flaccumfaciens pv. flaccumfaciens causing a bacterial tan spot of soybean in Russia.

The bacteria Pseudomonas savastanoi pv. glycinea (Coerper, 1919; Gardan et al., 1992) (Psg) and Curtobacterium flaccumfaciens pv. flaccumfaciens (Hedges 1922) (Cff) are harmful pathogens of soybean (Glycine max). Presently, there are several strategies to control these bacteria, and the usage of environmentally friendly approaches is encouraged. In this work, purified essential oils (EOs) from 19 plant species and total aqueous and ethanolic plant extracts (PEs) from 19 plant species were tested in vitro to observe their antimicrobial activity against Psg and Cff (by agar diffusion and broth microdilution method). Tested EOs and PEs produced significant bacterial growth inhibition with technologically acceptable MIC and MBC values. Non-phytotoxic concentrations for Chinese cinnamon and Oregano essential oils and leather bergenia ethanolic extract, which previously showed the lowest MBC values, were determined. Testing of these substances with artificial infection of soybean plants has shown that the essential oils of Chinese cinnamon and oregano have the maximum efficiency against Psg and Cff. Treatment of leaves and seeds previously infected with phytopathogens with these essential oils showed that the biological effectiveness of leaf treatments was 80.6-77.5% and 86.9-54.6%, respectively, for Psg and Cff. GC-MS and GC-FID analyzes showed that the major compounds were 5-Methyl-3-methylenedihydro-2(3H)-furanone (20.32%) in leather bergenia ethanolic extract, cinnamaldehyde (84.25%) in Chinese cinnamon essential oil and carvacrol (62.32%) in oregano essential oil.

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.

Plant growth promoting (PGP) bacteria are known to enhance plant growth and protect them from environmental stresses through different pathways. The rhizosphere of perennial plants, including olive, may represent a relevant reservoir of PGP bacteria. Here, seven bacterial strains isolated from olive rhizosphere have been characterized taxonomically by 16S sequencing and biochemically, to evaluate their PGP potential. Most strains were identified as Pseudomonas or Bacillus spp., while the most promising ones belonged to genera Pseudomonas and Curtobacterium. Those strains have been tested for their capacity to grow under osmotic or salinity stress and to improve the germination and early development of Triticum durum subjected or not to those stresses. The selected strains had the ability to grow under severe stress, and a positive effect has been observed in non-stressed seedlings inoculated with one of the Pseudomonas strains, which showed promising characteristics that should be further evaluated. The biochemical and taxonomical characterization of bacterial strains isolated from different niches and the evaluation of their interaction with plants under varying conditions will help to increase our knowledge on PGP microorganisms and their use in agriculture.

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