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Abstract Background Bacterial contamination poses a high risk to the successful establishment and maintenance of plant tissue cultures. The aim of this study was to identify the isolates representing the frequent bacterial contaminants of Prunus rootstock tissue cultures and to determine the most effective concentration of nanomaterials for Curtobacterium sp. strain A7_M15 elimination without a negative impact on explants. Results Six Curtobacterium sp. strains were isolated and identified, and the whole-genome sequence was obtained for strain A7_M15. Two nanocomposites, reduced graphene oxide–copper–silver and silver–selenium, with the highest bactericidal activity were selected for elimination of Curtobacterium sp. contamination in Gisela 5 rootstock tissue cultures. Both nanocomposites showed 100% inhibition of bacterial plaque formation on culture medium at concentrations of 100, 200 and 400 mg L-1 Ag (2 ×–8 × MBC). The quantity of Curtobacterium sp. on culture medium assessed using cfu enumeration was reduced by 92% and 74% in comparison to the positive control after treatment with reduced graphene oxide–silver–copper and silver–selenium at a concentration of 200 mg L-1 Ag, respectively. None of the tested concentrations resulted in a decrease in Curtobacterium sp. quantity in explants. Curtobacterium sp. was detected in donor Gisela 5 plants, indicating an endophytic character of this bacterium. The dry weight of explants was not negatively affected by the application of nanocomposites regardless of concentration, and no detrimental effect of either nanocomposite at 100 or 200 mg L-1 Ag on the surface covered by plants was observed. Conclusions Reduced graphene oxide–silver–copper and silver–selenium nanocomposites at 200 mg L-1 Ag effectively limited the Curtobacterium sp. presence in micropropagated Prunus rootstock without causing phytotoxicity; therefore, those treatments could be offered as prevention with a high activity against bacterial contamination in plant tissue cultures. Graphical Abstract

There exists a wide range of plant pathogens that are commonly referred to as seed-borne pathogens due to their dominant mode of spread. Treating seeds to eliminate such pathogens is therefore very important in contemporary seed production. In the present study, eight types of nanoparticles were evaluated for their effectiveness against Xanthomonas hortorum pv. carotae, a seed-borne pathogen that affects plants of the Apiaceae family. Initially, parameters considering the inhibitory and bactericidal activity of individual nanoparticles were evaluated under in vitro conditions. In this way, three nanoparticles based on copper, silver, and silver/selenium composite were identified as being the most effective. Subsequently, their ability to eliminate Xanthomonas hortorum pv. carotae from artificially infected carrot seeds was tested. This was achieved through the qPCR quantification of the pathogen in 14-day-old plantlets developed from seeds inoculated with Xhc. Based on the obtained results, copper-based nanoparticles were the most effective, resulting in an approximately 10-fold decrease in the occurrence of Xhc in plantlets compared to the untreated control. Taking into account the fact that X. hortorum pathovars also attack other important horticultural crops, the presented results may have a much wider scope than just carrot seeds.

The seed-borne bacterium Xanthomonas campestris pv. campestris (Xcc) as a causal organism of black rot disease remains the most serious bacterial problem of agricultural production of cruciferous plants worldwide. The eradication of a primary inoculum originating in seeds is available, but no treatment is totally effective. With the threat of developing chemical resistance and increasing pressure for sustainable disease management, biocontrol methods represent one of the main strategies currently applied in agriculture. Natural antimicrobials, including essential oils, are promising tools in disease management with low risks of environmental pollution and impact on human health. Thyme and clove essential oils were demonstrated to be highly effective in Xanthomonas studies in vitro; therefore, their application in black rot control was evaluated in this study. From five phenolic substances originating from thyme and clove essential oils (carvacrol, eugenol, linalool, p-cymene and thymol), the most promising in vitro results were observed with carvacrol, for which 0.0195% led to the death of all Xcc cells in 30 min. Moreover, a synergistic antibacterial effect of carvacrol and thymol solutions decreased the minimal inhibition concentration to 0.0049% and 0.0195% for carvacrol and thymol, respectively. Using the quadruple bactericidal values, the complete elimination of Xcc from the surface of infested cabbage seeds was obtained for both carvacrol and thymol solutions and their combined mixture at 2 MIC value. The elimination of bacterial infection from germinated cabbage plants was observed for both plate counting and quantitative real-time PCR methods. We also evaluated the effect of the application of phenolic treatment on the seed germination and germinated plants. Our results suggest a high potential of the application of carvacrol and thymol in vegetable seed production, specifically for cabbage, thus representing a suitable alternative to cupric derivatives.

Phytophthora castaneae, an oomycete pathogen causing root and trunk rot of different tree species in Asia, was shown to harbor a rich diversity of novel viruses from different families. Four P. castaneae isolates collected from Chamaecyparis hodginsii in a semi-natural montane forest site in Vietnam were investigated for viral presence by traditional and next-generation sequencing (NGS) techniques, i.e., double-stranded RNA (dsRNA) extraction and high-throughput sequencing (HTS) of small RNAs (sRNAs) and total RNA. Genome organization, sequence similarity, and phylogenetic analyses indicated that the viruses were related to members of the order Bunyavirales and families Endornaviridae, Megabirnaviridae, Narnaviridae, Totiviridae, and the proposed family “Fusagraviridae.” The study describes six novel viruses: Phytophthora castaneae RNA virus 1–5 (PcaRV1-5) and Phytophthora castaneae negative-stranded RNA virus 1 (PcaNSRV1). All six viruses were detected by sRNA sequencing, which demonstrates an active RNA interference (RNAi) system targeting viruses in P. castaneae. To our knowledge, this is the first report of viruses in P. castaneae and the whole Phytophthora major Clade 5, as well as of the activity of an RNAi mechanism targeting viral genomes among Clade 5 species. PcaRV1 is the first megabirnavirus described in oomycetes and the genus Phytophthora.

Symbiotic interaction between plants and microorganisms in the rhizosphere is an important factor affecting plant growth and fitness. Arbuscular mycorrhiza fungi symbiosis increases resistance of the plants to stress factors, including pathogens. Tomato brown rugose fruit virus (ToBRFV) is an important destructive virus damaging tomatoes and peppers with losses that can reach 100%. It is listed on the list of current quarantine organisms in the Czech Republic. The aim of this study was to evaluate influence of root colonization with Funneliformis mosseae or/and Azospirillum brasilense on ToBRFV symptoms and viral titre reduction. Plants treated with arbuscular mycorhizal fungi (AMF) had lower symptom emergence after 14 dpi, however there was no difference in symptoms emergence after 21 dpi within all treatments. The highest colonization intensity by Funneliformis mosseae was detected in ToBRFV negative plants treated with both AMF and Azospirillum (AZO) and the lowest in ToBRFV positive plants with the same treatment (AMF + AZO). Colonization intensity of Azospirillum brasilense in all treated variants went from 20% to 41%. Results suggest that the combination of those two beneficial microorganisms in ToBRFV-infected plants negatively affected AMF colonization.

Grapevine trunk diseases (GTDs) pose a major threat to the wine industry worldwide. Currently, efficient biological methods or chemical compounds are not available for the treatment of infected grapevines. In the present study, we used an extract from the knotwood of spruce trees as a biological control against GTDs. Our in vitro trial was focused on the antifungal effects of the extract against the most common GTD pathogens—Cadophora luteo-olivacea, Dactylonectria torresensis, Diaporthe ampelina, Diaporthe bohemiae, Diplodia seriata, Eutypa lata, and Phaeoacremonium minimum. Our in vitro trial revealed a high antifungal effect of the extract against all tested fungi. The inhibition rates varied among the different species from 30% to 100% using 1 mg·mL−1 extract. Subsequently, the efficiency of the extract was supported by an in planta experiment. Commercial grafts of Vitis vinifera were treated with the extract and planted. The total genomic DNA of grapevines was extracted 10 days and 180 days after the treatment. The fungal microbial diversities of the treated/untreated plants were compared using high-throughput amplicon sequencing (HTAS). Treated plants showed 76.9% lower relative abundance of the genus Diaporthe and 70% lower relative abundance of the genus Phaeoacremonium 10 days after treatment. A similar scenario was observed for the genus Cadophora 180 days after treatment, where treated plants showed 76% lower relative abundance of this genus compared with untreated grapevines.

Grapevine Pinot gris virus (GPGV) is considered to be a causal agent of Grapevine Leaf Mottling and Deformation (GLMD) disease that has been reported worldwide through the grapevine-growing regions. Seven grapevines that were collected from a vineyard in the Czech Republic were tested for the presence of GPGV in leaf and phloem tissues. Each of the seven grapevines was infected by GPGV, from which sic symptoms were mostly shown without a typical mottling. The phylogeny based on RNA-dependent RNA polymerase and movement/coat protein sequences indicated the same origin of the GPGV isolates. The GPGV titer was the highest in the grapevines with the highest GLMD-like symptoms; however, some of the grapevines with milder GLMD-like symptoms had a lower GPGV titer than the asymptomatic grapevine. Soil analysis showed uneven boron content in the direct vicinity of the grapevines, while the boron content in the grapevines was more, even showing no boron deficiency. The quantitative analysis of selected gene expressions associated with boron efflux and transport only partially explained the boron content in the soil and grapevines and only in the grapevines growing in soils with the highest or lowest boron contents. The VvBor2 and VvNIP5 genes had a higher expression and VvNIP6 had a lower expression in the grapevine growing in the soil with the lowest boron content, while a low expression of VvBor1 and VvBor2 was observed in the grapevine that was grown in the soil with the highest boron content.

ABSTRACT In this study, we investigated the impact of zinc oxide (ZnO) and zinc oxide nanoparticles (ZnONPs) on Escherichia coli. These compounds are commonly used as fertilizers and feed additives and may have unintended consequences on bacteria. We conducted phenotypic and multi-omics analyses of E. coli exposed to sub-lethal concentrations of ZnO and ZnONPs for 40 sub-culturings and 20 sub-culturings with zinc followed by 20 sub-culturings without zinc in an attempt to reverse zinc effects. An extended treatment with ZnO for 40 sub-culturings had the greatest impact leading to bacterial resistance to aminoglycosides, cephalosporins, and sulfonamides while the treatment with ZnONPs for 40 sub-culturings led to the elevated MIC to chloramphenicol only. Cells exposed to these treatments were thicker and had retarded growth in elevated temperatures. Importantly, zinc withdrawal reversed most phenotypic changes. Zinc exposure caused dramatic changes in cell transcripts and proteins with a role in antibiotic response, heat stress, growth regulation, cell shape, and biofilm formation. An extended zinc exposure led to overall major alterations in E. coli biology and resulted in multi-drug resistance.IMPORTANCEA long-term exposure of bacteria to zinc oxide and zinc oxide nanoparticles leads to major alterations in bacterial morphology and physiology. These included biochemical and physiological processes promoting the emergence of strains with multi-drug resistance and virulence traits. After the removal of zinc pressure, bacterial phenotype reversed back to the original state; however, certain changes at the genomic, transcriptomic, and proteomic level remained. Why is this important? The extensive and intensive use of supplements in animal feed effects the intestinal microbiota of livestock and this may negatively impact the health of animals and people. Therefore, it is crucial to understand and monitor the impact of feed supplements on intestinal microorganisms in order to adequately assess and prevent potential health risks.

Phyllosticta ampelicida causes grapevine black rot, a potentially damaging disease for grape production. This paper reports the draft genome sequence of P. ampelicida PA1 Galicia CBS 148563, which is 30.55 Mb and encodes 10,691 predicted protein-coding genes. This is the first sequence genome assembly of P. ampelicida, and this information is a valuable resource to support genomic attributes for determining pathogenic behaviour and comparative genomic analyses of grapevine black rot fungi.

Tomato is the most consumed vegetable in the world. The tomato brown rugose fruit virus (ToBRFV) is an important destructive virus that damages tomatoes and peppers with significant economic impact. The detection and characterization of this important viral pathogen were evaluated at the molecular and morphological level. The viral isolate was purified and inoculated on tomato and pepper plants. Small RNAs were sequenced in both plants and the profiles were compared. The complete genome of the isolate was obtained, and microRNA (miRNA) profiles were unveiled by small RNA sequencing. Symptoms caused by the isolate were also described and the morphology of the isolate was observed by transmission electron microscopy. Our results contribute to further understanding of the role of miRNAs in ToBRFV pathogenesis, which may be crucial for understanding disease symptom development in tomatoes and peppers.