Iskanje po repozitoriju

A+ | A- | | SLO | ENG

Povzetek: Dickeya fangzhongdai, an aggressive plant pathogen, causes symptoms on a variety of crops and ornamental plants including bleeding canker of Asian pear trees. Historical findings stress the need for a specific detection tool for D. fangzhongdai to prevent overlooking the pathogen or assigning it to general Dickeya spp. Therefore, a qualitative real-time PCR for specific detection of D. fangzhongdai has been developed and validated. The developed assay shows selectivity of 100%, diagnostic sensitivity of 76% and limit of detection with 95% confidence interval in plant matrices ranging from 311 to 2,275 cells/mL of plant extracts. The assay was successfully used in a retrospective survey of selected host plants of relevance to Europe and environmental niches relevant to D. fangzhongdai. Samples of potato tubers and plants, plants from the Malinae subtribe (apple, pear, quince, and Asian pear tree) and fresh surface water from Slovenia were analyzed. D. fangzhongdai was not detected in any plant samples, however, 12% of surface water samples were found to be positive.
Ključne besede: molecular testing, diagnostics, plant pathogen, real-time PCR, Dickeya, survey, water
Objavljeno v DiRROS: 07.08.2024; Ogledov: 54; Prenosov: 59
Celotno besedilo (1,94 MB)
Gradivo ima več datotek! Več...

Povzetek: Background Detection and quantification of plant pathogens in the presence of inhibitory substances can be a challenge especially with plant and environmental samples. Real-time quantitative PCR has enabled high-throughput detection and quantification of pathogens; however, its quantitative use is linked to standardized reference materials, and its sensitivity to inhibitors can lead to lower quantification accuracy. Droplet digital PCR has been proposed as a method to overcome these drawbacks. Its absolute quantification does not rely on standards and its tolerance to inhibitors has been demonstrated mostly in clinical samples. Such features would be of great use in agricultural and environmental fields, therefore our study compared the performance of droplet digital PCR method when challenged with inhibitors common to plant and environmental samples and compared it with quantitative PCR. Results Transfer of an existing Pepper mild mottle virus assay from reverse transcription real-time quantitative PCR to reverse transcription droplet digital PCR was straight forward. When challenged with complex matrices (seeds, plants, soil, wastewater) and selected purified inhibitors droplet digital PCR showed higher resilience to inhibition for the quantification of an RNA virus (Pepper mild mottle virus), compared to reverse transcription real-time quantitative PCR. Conclusions This study confirms the improved detection and quantification of the PMMoV RT-ddPCR in the presence of inhibitors that are commonly found in samples of seeds, plant material, soil, and wastewater. Together with absolute quantification, independent of standard reference materials, this makes droplet digital PCR a valuable tool for detection and quantification of pathogens in inhibition prone samples.
Ključne besede: PCR amplification, inhibition, qPCR, droplet digital PCR, environmental samples
Objavljeno v DiRROS: 02.08.2024; Ogledov: 84; Prenosov: 55
Celotno besedilo (807,79 KB)
Gradivo ima več datotek! Več...

Povzetek: The presence of plastics in the environment is currently one of the most pressing global environmental problems. Microorganisms start to form biofilms on plastic surfaces when they first come in contact with the biosphere; however, these interactions and processes are little understood, especially in freshwaters. This study aimed to better understand the colonization process of microorganisms from activated sludge on plastic materials exhibiting different surface characteristics. We inoculated synthetic fabric (PET), water bottles (PET), and plastic bags for packing vegetables and fruits (HDPE) with microorganisms from activated sludge. Mixtures of plastics and activated sludge, as well as the control, were incubated at 22-24°C in Bushnell Haas (BH) liquid medium and shaken at 120 rpm for two months. The mixtures were sub-sampled weekly and seeded into fresh BH medium with test plastic materials to avoid feeding microorganisms on dead biomass. The colonization was followed by measuring optical density (OD600) of liquid medium, by measurements of isotopic composition of carbon (δ13C) in untreated and treated plastic materials and, with in-specting the plastics surface with scanning electron microscopy (SEM). Overall, the study confirmed differences between colonizing microorganisms on different plastic material when comparing SEM micrographs of materials from the flasks inoculated with activated sludge. The texture of the HDPE bag changed during the experiment in both, control and inoculated flasks, but it is not clear whether the observed changes were due to abiotic or biotic factors. We concluded that microorganisms from activated sludge are capable of colonizing both PET and HDPE materials, and biofilm formation is most probably influenced by the chemical composition of plastics and their surface characteristics.
Ključne besede: biofilm, plastics, SEM, isotopic composition of carbon, co-cultivation, UV sterilization
Objavljeno v DiRROS: 31.07.2024; Ogledov: 82; Prenosov: 67
Celotno besedilo (678,88 KB)
Gradivo ima več datotek! Več...

Povzetek: Accurate and precise nucleic-acid quantification is crucial for clinical and diagnostic decisions, as overestimation or underestimation can lead to misguided treatment of a disease or incorrect labelling of the products. Digital PCR is one of the best tools for absolute nucleic-acid copy-number determination. However, digital PCR needs to be well characterised in terms of accuracy and sources of uncertainty. With droplet digital PCR, discrepancies between the droplet volume assigned by the manufacturer and measured by independent laboratories have already been shown in previous studies. In the present study, we report on the results of an inter-laboratory comparison of different methods for droplet volume determination that is based on optical microscopy imaging and is traceable to the International System of Units. This comparison was conducted on the same DNA material, with the examination of the influence of parameters such as droplet generators, supermixes, operators, inter-cartridge and intra-cartridge variability, and droplet measuring protocol. The mean droplet volume was measured using a QX200™ AutoDG™ Droplet Digital™ PCR system and two QX100™ Droplet Digital™ PCR systems. The data show significant volume differences between these two systems, as well as significant differences in volume when different supermixes are used. We also show that both of these droplet generator systems produce droplets with significantly lower droplet volumes (13.1%, 15.9%, respectively) than stated by the manufacturer and previously measured by other laboratories. This indicates that to ensure precise quantification, the droplet volumes should be assessed for each system.
Ključne besede: droplet digital PCR, droplet volume, DNA quantification, optical microscopy imaging
Objavljeno v DiRROS: 25.07.2024; Ogledov: 120; Prenosov: 78
Celotno besedilo (565,33 KB)
Gradivo ima več datotek! Več...

Povzetek: Background The Dickeya genus is part of the Pectobacteriaceae family that is included in the newly described enterobacterales order. It comprises a group of aggressive soft rot pathogens with wide geographic distribution and host range. Among them, the new Dickeya fangzhongdai species groups causative agents of maceration-associated diseases that impact a wide variety of crops and ornamentals. It affects mainly monocot plants, but D. fangzhongdai strains have also been isolated from pear trees and water sources. Here, we analysed which genetic novelty exists in this new species, what are the D. fangzhongdai-specific traits and what is the intra-specific diversity. Results The genomes of eight D. fangzhongdai strains isolated from diverse environments were compared to 31 genomes of strains belonging to other Dickeya species. The D. fangzhongdai core genome regroups approximately 3500 common genes, including most genes that encode virulence factors and regulators characterised in the D. dadantii 3937 model strain. Only 38 genes are present in D. fangzhongdai and absent in all other Dickeyas. One of them encodes a pectate lyase of the PL10 family of polysaccharide lyases that is found only in a few bacteria from the plant environment, soil or human gut. Other D. fangzhongdai-specific genes with a known or predicted function are involved in regulation or metabolism. The intra-species diversity analysis revealed that seven of the studied D. fangzhongdai strains were grouped into two distinct clades. Each clade possesses a pool of 100–150 genes that are shared by the clade members, but absent from the other D. fangzhongdai strains and several of these genes are clustered into genomic regions. At the strain level, diversity resides mainly in the arsenal of T5SS- and T6SS-related toxin-antitoxin systems and in secondary metabolite biogenesis pathways. Conclusion This study identified the genome-specific traits of the new D. fangzhongdai species and highlighted the intra-species diversity of this species. This diversity encompasses secondary metabolites biosynthetic pathways and toxins or the repertoire of genes of extrachromosomal origin. We however didn’t find any relationship between gene content and phenotypic differences or sharing of environmental habitats. Background Soft rot Pectobacteriaceae are Enterobacterales responsible for considerable economic losses in several important crops and ornamental plants [1,2,3]. Their virulence is mainly due to the production and secretion of a battery of plant cell wall degrading enzymes (PCWDEs) that cause maceration of the plant tissue; however, several other virulence factors have also been characterized [2, 4]. These bacteria often exhibit a very broad host range, and recent outbreaks in potato, for example, resulted from the action of a cohort of bacteria belonging to different Pectobacteriaceae species in a complex population dynamics history [5]. The Pectobacteriaceae family includes two genera comprising soft rot bacteria, Pectobacterium and Dickeya. The Dickeya genus was formed in 2005 by the reclassification of former Erwinia chrysanthemi into six species [6]. It has recently undergone multiple phylogenetic changes, including the addition of three new species, Dickeya solani [7], Dickeya aquatica [8] and, more recently, Dickeya fangzhongdai [9]. The description of this last new species was based on three isolates from pear trees in China with bleeding canker necrosis [9], but it was extended by a large number of strains isolated from monocot plants from Japan [10, 11]. D. fangzhongdai strains were associated with soft rot symptoms of many ornamental and economically important staple food plants [10, 12, 13], thereby highlighting the broad host range of the species. While there is little information regarding associated economic damages and the extent of its occurrence in different host plants outside of Asia, Alič et al. [14] recently identified D. fangzhongdai as the causative agent of soft rot of orchids in commercial production in Europe, starting with material from Asia [11]. Moreover, as previously reported, bacteriophages of different families, and active against D. fangzhongdai, were isolated from a wastewater treatment plant not associated to the orchid production site. This would suggest that D. fangzhongdai bacteria may be more widespread in nature than could currently be concluded on the basis of symptoms in plants. Its occurrence in water would suggest that it may potentially have a wider ecological niche than genomically close Dickeya spp., that is, Dickeya dadantii, Dickeya dianthicola, and D. solani. Previous experience with D. solani has shown that novel species or isolates can lead to clonal spread and high losses in affected host plants [15]. Together with repeated introductions of D. fangzhongdai, the co-occurrence of genetically and phenotypically diverse strains on the same plants (e.g., B16 and S1 on orchids, as reported by Alič et al. [11]) increases the probability of the development of recombined strains with novel pathogenic potential and may present a risk to agriculturally important plants. Their aggressiveness, high maceration potential on various plant tissues, and persistence in potato plants further exacerbate the risk for agriculture. Therefore, in this paper, we analysed the genomic characteristics of the D. fangzhongdai species, compared it to the other Dickeya species and determine the inter- and intra- species diversity. The study addressed the question whether the presence of the isolates in a specific environment is associated to a specific set of genes (water vs plant symptoms, monocots vs dicots, different geographical origin). We also analysed the virulence gene arsenal, in order to evaluate the virulence potential of this species. Methods Dickeya strain selection All D. fangzhongdai genomes publicly available in the NCBI database were included in this study. These genomes were compared to five D. solani, four D. dadantii, five D. dianthicola, five D. chrysanthemi, seven D. zeae, one D. aquatica, two D. paradisiaca and two unassigned Dickeya genomes extracted from the NCBI database. Information on the provenance and genomic data of the D. fangzhongdai strains used in this study are summarized in Table 1. The accession numbers and phylogenetic position of the other Dickeya strains used for the SiLix analyses are presented in Additional file 1: Figure S1.
Ključne besede: T5SS, T6SS, NRPS/PKS, zeamine, oocydin A, plant-bacteria interactions, plasmid, Dickeya fangzhongdai
Objavljeno v DiRROS: 23.07.2024; Ogledov: 99; Prenosov: 82
Celotno besedilo (2,95 MB)
Gradivo ima več datotek! Več...