Search Results (219)

CRISPR/Cas12a systems coupled with lateral flow tests (LFTs) are a promising route to rapid, instrument-free nucleic acid diagnostics due to conversion target recognition into a simple visual readout via cleavage of dual-labeled single-stranded DNA reporters. However, the conventional CRISPR/Cas12a–LFT system is constructed in a format where the intact reporter should block nanoparticle conjugate migration and can produce false-positive signals and shows strong dependence on component stoichiometry and kinetics. Here, we present the first combined experimental and theoretical analysis quantifying these limitations and defining practical solutions. The experimental evaluation included 480 variants of LFT configuration with reporters differing in the concentration of interacting components and the kinetic conditions of the interactions. The most influential factor leading to 100% false-positive results was insufficient interaction time between the components; pre-incubation of the conjugate with the reporter for 5 min eliminated these artifacts. Theoretical analysis of the LFT kinetics based on a mathematical model confirmed kinetic constraints at interaction times below a few minutes, which affect the detectable signal. Reporter concentration and conjugate architecture represented the second major factors: lowering reporter concentration to 20 nM and using smaller gold nanoparticles with multivalent fluorescent reporters markedly improved sensitivity. The difference in sensitivity between various LFT configurations exceeded 50-fold. The combination of identified strategies eliminated false-positive reactions and enabled the detection of up to 20 pM of DNA target (the hisZ gene of Erwinia amylovora, a bacterial phytopathogen). The strategies reported here are general and readily transferable to other DNA targets and CRISPR/Cas12a amplification-free diagnostics. Full article

Fire blight, caused by Erwinia amylovora, is one of the most damaging bacterial diseases affecting apple production and the safety of wild Malus sieversii populations in Central Asia. Effective monitoring relies on accurate molecular diagnostics; however, comparative data on commonly used detection methods remain limited for the region. In this study, we evaluated the performance of three molecular assays—LAMP, real-time PCR, and targeted nanopore sequencing of a 16S rRNA gene fragment—using 124 plant samples exhibiting fire blight symptoms collected from 30 sites across Southern Kazakhstan and Kyrgyzstan. The results of LAMP, real-time PCR, and the amplification of 16S sequences were highly consistent with each other. Targeted 16S nanopore sequencing reliably identified E. amylovora in all PCR-positive samples, yielding high read counts and consistent species-level classification, although the analyzed 16S region provided limited resolution for intraspecies variation. Across sampling locations, abandoned orchards represented major reservoirs of infection compared to maintained orchards and wild populations. Our results confirm that all three approaches are robust tools for detecting E. amylovora. These findings support the importance of different molecular diagnostic methods to assist fire blight surveillance in the region. Full article

Priestia megaterium is a maize endophyte that may help the plant defend itself against bacterial and fungal pathogens. This study aimed to identify antimicrobials produced by two P. megaterium endophytes (FS10 and FS11) from maize and determine if seed coating with either strain could increase resistance to pathogens. Volatiles emitted by both isolates reduced the hyphal growth of fungi by 17–76%. Gas chromatography analysis found that each strain emitted isovaleric acid (IVA) and 3-methyl-1-butanol (3MB). Volatiles produced by each isolate inhibited bacterial growth, especially Clavibacter michiganensis ssp. michiganensis (Cmm). IVA killed all Cmm cells at 208 µL L⁻¹, while 3MB inhibited Cmm growth by 51% at 208 µL L⁻¹. Diluted cell-free extracts from FS10 and FS11 cultures stopped growth of Cmm, Erwinia amylovora and Ustilago maydis but did not arrest growth of Fusarium verticillioides. The treatment of corn seeds with FS10 or FS11 reduced leaf damage by 38–84% in young plants caused by Bipolaris maydis, Colletotrichum graminicola (Ces.) G.W. Wilson 1914, Exserohilum turcicum and Pythium sylvaticum. FS10 and FS11 isolates exuded volatile and soluble compounds that were more effective in slowing growth of bacteria than fungi. It is likely that corn seed treatment with FS10 and FS11 triggers induced systemic resistance, which mitigates leaf damage caused by maize pathogens. Full article

The stem blight disease on Elaeocarpus spp. was newly identified in 2022 on E. rugosus and E. hain in Guangzhou, representing a serious stem disease that causes branch rot and cankers in Elaeocarpus species and can lead to whole-plant death in severe cases. Strain ZX-13 was isolated from Elaeocarpus rugosus stems and showed excellent antagonism against the Elaeocarpus stem blight pathogen Pseudocryphonectria elaeocarpicola. Based on morphological characteristics, physiological and biochemical results, and molecular biology analyses, the strain was identified as Erwinia phyllosphaerae. Whole-genome sequencing and annotation indicate that strain ZX-13 has a high-quality draft assembly of 4,686,433 bp, with a GC content of 53.85%, encoding 4189 genes, 86 tRNA, 22 rRNA, and 110 ncRNA. Eight antifungal enzymes from the GH family may be crucial factors to its antagonistic activity. Using antiSMASH 7.1.0, the ZX-13 genome predicted eight BGCs (RiPPs, NRPs, terpenes, etc.), with one showing 100% similarity to carotenoid biosynthesis. Novel candidates include an O-antigen, hassallidin C, and lankacidin C. The study identified Erwinia phyllosphaerae ZX-13 as an antagonistic bacterial strain for the first time, indicating substantial potential for biocontrol applications. Full article

Bacterial diseases of forest trees represent an increasing threat to ecosystem health and the sustainability and resilience of forest management, particularly under changing climate conditions. One of the key yet still insufficiently understood adaptive mechanisms of pathogens is biofilm formation—a structured community of bacterial cells embedded in a matrix of extracellular polymeric substances (EPS), which provides protection against stress factors, biocides, and the host’s defensive responses such as antimicrobial compounds or immune reactions. This paper presents a comprehensive review of current knowledge on the role of biofilms in the bacterial pathogenesis of forest trees, covering their formation mechanisms, molecular regulation, and ecological significance. Four key stages of biofilm development are discussed—adhesion, microcolony formation, EPS production, and dispersion—along with the roles of quorum sensing systems and c-di-GMP-based signaling in regulating these processes. Examples of major tree pathogens are presented, including Pseudomonas syringae, Erwinia amylovora, Xylella fastidiosa, the Brenneria–Gibbsiella complex associated with Acute Oak Decline (AOD) and Lonsdalea populi. Biofilm formation is shown to play a crucial role in the colonization of xylem, leaf surfaces, and tissues undergoing necrosis, where biofilms may stabilize decomposition zones and support saprophytic–pathogenic transitions. In the applied section, the concept of “biofilm-targeted control” is discussed, encompassing both chemical and biological strategies for disrupting biofilm structure—from quorum-sensing inhibitors and EPS-degrading enzymes to the use of biosurfactants and antagonistic microorganisms. The need for in situ research in forest environments and the adaptation of advanced imaging (CLSM, micro-CT) and metagenomic analyses to tree systems is also emphasized. This review concludes that biofilms are not merely a physiological form of bacterial organization but a complex adaptive system essential for the survival and virulence of pathogens in forest ecosystems. Understanding their functions is fundamental for developing sustainable and ecologically safe phytosanitary strategies for forest protection. Full article

Plants exposed to combined abiotic and biotic stresses often exhibit complex physiological responses that cannot be predicted from single stress factors. In this study, we evaluated the interactive effects of temperature stress and Erwinia amylovora infection on pear (Pyrus pyrifolia) leaves under five temperature conditions (10, 15, 25, 30, and 35 °C) with or without pathogen inoculation, using chlorophyll fluorescence analysis and RGB imaging over a 7-day period. Photosynthetic performance remained optimal at 25 °C under single temperature conditions, whereas pathogen inoculation alone caused PSII damage and reduced energy dissipation. Under combined stress, PSII responses exhibited temperature-dependent patterns: at 10, 15 °C, photoprotective mechanisms were partially maintained; at 25, 30 °C, severe structural and functional damage occurred; and at 35 °C, pathogen activity was suppressed while partial recovery of PSII was observed. By integrating chlorophyll fluorescence analysis with a linear mixed-effect model (LMM), distinct patterns of sensitivity were identified among fluorescence parameters, with ΦNO responding to single stress factors, and Fm, Fv, Fp, Fv/Fo, and qL showing significant three-way interactions. These findings highlight temperature-dependent strategies of pear leaves to cope with fire blight and emphasize the utility of chlorophyll fluorescence analysis for evaluating photosynthetic resilience. From an applied perspective, chlorophyll fluorescence could serve as a rapid, non-destructive tool for screening pear cultivars with enhanced tolerance to bacterial fire blight, contributing to more efficient orchard management strategies. Full article

Phage therapy has garnered significant attention due to the rise of life-threatening multidrug-resistant pathogenic bacteria and the growing awareness of the transfer of resistance genes between pathogens. Considering this, phage therapy applications are being extended to target plant pathogenic bacteria, such as Erwinia amylovora, which causes fire blight in apple and pear orchards. Understanding the mechanisms involved in phage resistance is crucial for enhancing the effectiveness of phage therapy. Despite the challenges of naturally developing a bacteriophage-insensitive mutant (BIM) of E. amylovora (without traditional mutagenesis methods), this study successfully created a BIM against the podovirus ϕEa46-1-A1. The parent strain, E. amylovora D7, and the BIM B6-2 were extensively compared at genomic, transcriptomic, and phenotypic levels. The phenotypic comparison included the metabolic behavior, biofilm formation, and in planta evaluations of pathogenicity. The results revealed a mutation in strain B6-2 in the rcsB gene, which encodes a second regulator in the Rcs two-component phosphorelay system (TCS). This mutation resulted in significant changes in the B6-2 BIM, including downregulation of amylovoran gene expression (e.g., an average log₂ fold change of −4.35 across amsA-L), visible alterations in biofilm formation, increased sensitivity to antibiotics (22.4% more sensitive to streptomycin), and a loss of pathogenicity as assessed in an apple seedling virulence model in comparison to the wildtype strain. The findings presented in this study highlight the critical role of the Rcs phosphorelay system in phage resistance in E. amylovora. Based on these findings, we have proposed a model that explains the effect of the B6-2 rcsB mutation on the Rcs phosphorelay system and its contribution to the development of phage resistance in E. amylovora. Full article

Needle cast (Lophodermium seditiosum Minter, Staley & Millar) in Scots pine (Pinus sylvestris L.) and European ash (Fraxinus excelsior L.) dieback (Hymenoscyphus fraxineus (T. Kowalski) Baral, Queloz & Hosoya) are among the most destructive forest and tree plantation diseases in Europe, threatening not only targeted plant species but also the whole ecosystem. While considerable research effort has focused on microbial antagonists against ash dieback, comparable investigations into needle cast biocontrol remain virtually absent from the literature. Here, isolated microbial antagonists from European ash and Scots pine were evaluated for their efficacy against respective pathogens. In vitro dual-culture assays revealed bacteria with strong inhibitory effects on pathogen growth, as well as multiple plant growth-promoting traits (PGPTs). It was found that bacteria from the genera of Pantoea, Erwinia, Priestia, and Pseudomonas inhibited the growth of H. fraxineus by ≥70%. Most significantly, our investigation revealed that bacteria isolated from Scots pine, belonging to the genera Pseudomonas, Bacillus, and Priestia, inhibited the growth of L. seditiosum by 50% to 80%, representing one of the first reported bacterial antagonisms for this neglected pathogen. All isolates were positive for at least two PGPTs, primarily due to mineralization of organic phosphate and the production of siderophores. The dual functional traits of isolated bacteria highlight their potential application in integrated forest protection strategies, particularly for the previously overlooked L. seditiosum pathosystem. Full article

Fire blight, caused by Erwinia amylovora, is a severe disease impacting pome fruit production worldwide, including in Saudi Arabia. This study evaluated antibiotic sensitivity and the potential of chemical and elicitor treatments to suppress E. amylovora isolates collected from various regions in Saudi Arabia. In the in vitro assays, at low antibiotic levels (10 µg/mL streptomycin and 25 µg/mL oxytetracycline), all Saudi Arabian strains exhibited minimal inhibition (zones ≤ 14 mm). Two isolates displayed partial tolerance at an intermediate oxytetracycline concentration (50 µg/mL). True sensitivity (zones > 18 mm) was mainly observed at the highest tested oxytetracycline dose (100 µg/mL). Regarding copper sulfate, all isolates showed no inhibition between 0.02 and 0.08 mM, while all isolates exhibited intermediate susceptibility at 0.16 mM. The second experimental phase examined in planta effects of streptomycin, salicylic acid (SA), and their combination on disease development in artificially inoculated apple (Malus domestica) shoots under greenhouse conditions. Both streptomycin and SA significantly reduced fire blight incidence (by 75%) and symptom severity, while the combined treatment yielded the greatest reduction in shoot necrosis and bacterial load. This is the first report demonstrating that SA, particularly when used in combination with streptomycin, can effectively suppress fire blight in Saudi Arabia. These results stress the importance of integrating resistance inducers into fire blight management strategies to counter the rise in antimicrobial resistance. Full article

We report a case of catheter-associated bloodstream infection caused by a putative novel species in the genus Erwinia, identified using whole-genome sequencing (WGS). A female adolescent receiving long-term home parenteral nutrition via a central venous catheter (CVC) presented with a fever. Gram-negative rods were isolated from two CVC-derived blood culture sets, while peripheral cultures remained negative. Conventional identification methods, including VITEK 2, Phoenix M50, MALDI-TOF MS, and 16S rRNA and rpoB gene sequencing, failed to achieve species-level identification. WGS was performed on the isolate using Illumina MiSeq. Genomic analysis revealed a genome size of 5.39 Mb with 56.8% GC content and high assembly completeness. The highest average nucleotide identity (ANI) was 90.3% with Pantoea coffeiphila, and ≤85% with known Erwinia species, suggesting that it represents a distinct taxon. Phylogenetic analyses placed the isolate within the Erwinia clade but separate from any known species. Antimicrobial susceptibility testing showed broad susceptibility. This case highlights the utility of WGS for the identification of rare or novel organisms not captured by conventional methods and expands the clinical spectrum of Erwinia species. While the criteria for species delineation were met, the phenotypic characterization remains insufficient to formally propose a new species. Full article

This study demonstrated that application of the particular plant growth-promoting rhizobacteria (PGPR) strains Erwinia sp. and Serratia sp. (named C15 and C20, respectively) significantly enhanced tea plant resilience in Zn (zinc)-, Pb (lead)-, and Zn + Pb-contaminated soils by the improving survival rates (over 60%) and chlorophyll content of tea plants, and by reducing the accumulation of these metals in tea plants’ tissues (by 19–37%). The PGPRs elevated key soil nutrients organic carbon (OC), total nitrogen (TH), hydrolysable nitrogen (HN), and available potassium (APO) and phosphorus (APH) contents. Compared to non-PGPR controls, both strains consistently increased microbial α-diversity (Chao1 index: +28–42% in Zn/Pb soils; Shannon index: +19–33%) across all contamination regimes. PCoA/UniFrac analyses confirmed distinct clustering of PGPR-treated communities, with strain-specific enrichment of metal-adapted taxa, including Pseudomonas (LDA = 6) and Bacillus (LDA = 4) under Zn stress; Rhodanobacter (LDA = 4) under Pb stress; and Lysobacter (LDA = 5) in Zn + Pb co-contamination. Fungal restructuring featured elevated Mortierella (LDA = 6) in Zn soils and stress-tolerant Ascomycota dominance in co-contaminated soils. Multivariate correlations revealed that the PGPR-produced auxin was positively correlated with soil carbon dynamics and Mortierellomycota abundance (r = 0.729), while the chlorophyll content in leaves was closely associated with Cyanobacteria and reduced by Pb accumulation. These findings highlighted that PGPR could mediate and improve in tea plant physiology, soil fertility, and stress-adapted microbiome recruitment under heavy metal contaminated soil and stress. Full article

Genomic islands (GIs) including integrative and conjugative elements (ICEs), prophages, and integrative plasmids are central drivers of horizontal gene transfer in bacterial plant pathogens. These elements often carry cargo genes encoding virulence factors, antibiotic and metal resistance determinants, and metabolic functions that enhance environmental adaptability. In plant-pathogenic species such as Pseudomonas syringae, GIs contribute to host specificity, immune evasion, and the emergence of novel pathogenic variants. ICEclc and its homologs represent integrative and mobilizable elements whose tightly regulated excision and transfer are driven by a specialized transcriptional cascade, while ICEs in P. syringae highlight the ecological impact of cargo genes on pathogen virulence and fitness. Pathogenicity islands further modulate virulence gene expression in response to in planta stimuli. Beyond P. syringae, GIs in genera such as Erwinia, Pectobacterium, and Ralstonia underpin critical traits like toxin biosynthesis, secretion system acquisition, and topoisomerase-mediated stability. Leveraging high-throughput genomics and structural biology will be essential to dissect GI regulation and develop targeted interventions to curb disease spread. This review synthesizes the current understanding of GIs in plant-pathogenic gammaproteobacteria and outlines future research priorities for translating mechanistic insights into sustainable disease control strategies. Full article

Dysbiosis is a strategy to control insect pests through disrupting symbiotic bacteria essential for their life cycle. The olive fly, Bactrocera oleae, has been considered a suitable system for dysbiosis, as the insect is strictly dependent on its unique symbiont Candidatus Erwinia dacicola. Here, we review older and recent results from studies of the interaction of the symbiont and its host fly. We then discuss possible methods for disrupting the symbiosis as a means to control the fly. Specifically, we summarize studies using microscopy methods that have investigated in great detail the organs where the bacterium resides and it is always extracellular. Furthermore, we discuss how genome sequences of both host and bacterium can provide valuable resources for understanding the interaction and transcriptomic analyses that have revealed important insights that can be exploited for dysbiosis strategies. We also assess experiments where compounds have been tested against the symbiont. The hitherto limited efficacy in decreasing bacterial abundance suggests that novel molecules and/or new ways for the delivery of agents will be important for successful dysbiosis strategies. Finally, we discuss how gene drive methods could be implemented in olive fly control, though a number of hurdles would need to be overcome. Full article

The market for non-concentrated (NFC) orange juice is increasing rapidly due to consumer demand for nutrients and flavor. However, it encounters challenges in microbial safety, particularly from Salmonella enterica and Listeria monocytogenes. This study aimed to exploit a bio-preservative for NFC orange juice. Results showed that the cyclic peptide cyclo-zp80r had good antibacterial activity, with minimum inhibitory concentration values of 2–8 μM against S. enterica and L. monocytogenes. It exhibited bactericidal action against S. enterica and bacteriostatic action against L. monocytogenes at a concentration of 128 μM. This study explored the effect of cyclo-zp80r on the pathogenicity of S. enterica and L. monocytogenes. The mortality rate of Galleria mellonella exposed to these pathogens in NFC orange juice decreased from 100% to 60% after cyclo-zp80r treatment, surpassing the effectiveness of nisin. Cyclo-zp80r exhibited depolarization effects on S. enterica and L. monocytogenes. It increased outer membrane permeability and damaged the membrane structure of S. enterica. Cyclo-zp80r also caused distinct morphological changes, mainly cell collapse in S. enterica and localized bubble-like protrusions in L. monocytogenes. It induced reactive oxygen species production and DNA binding. The species diversity and abundance in NFC orange juice were also reduced by cyclo-zp80r, particularly in the genera Pantoea, Aeromonas, Pseudomonas, and Erwinia. Additionally, cyclo-zp80r exhibited excellent stability at high temperature (121 °C, 5 min) and in fresh orange juice. These results suggest that cyclo-zp80r could be developed as an effective food bio-preservative. Full article

L-asparaginase (L-ASNase) remains a vital chemotherapeutic agent for acute lymphoblastic leukemia (ALL), primarily due to its mechanism of depleting circulating asparagine essential for leukemic cell proliferation. However, existing ASNases (including pegylated ones) face limitations including immunogenicity, rapid clearance, and off-target toxicities. Earlier, we have shown that the conjugation of L-ASNase with the polyamines and their copolymers results in significant enhancement of the antiproliferative activity due to accumulation in tumor cells. We suggested that this effect is probably mediated by polyamine transport system (PTS) receptors that are overexpressed in ALL cells. Here, we investigated the effect of competitive inhibitors of PTS receptors to the L-ASNase interaction with cancer cells (L5178Y, K562 and A549). L-ASNase from Rhodospirillum rubrum (RrA), Erwinia carotovora (EwA), and Escherichia coli (EcA) were conjugated with natural polyamines (spermine—spm, spermidine—spd, putrescine—put) and a synthetic branched polymer, polyethyleneimine 2 kDa (PEI2 ), using carbodiimide chemistry. Polyamine conjugation with L-ASNase significantly increased enzyme binding and cellular uptake, as quantified by fluorimetry and confocal microscopy. This increased cellular uptake translated into increased cytotoxicity of L-ASNase conjugates. The presence of competitive ligands to PTS receptors decreased the uptake of polyamine-conjugated enzymes-fatty acid derivatives of polyamines produced the strongest suppression. Simultaneously with this suppression, in some cases, competitive ligands to PTS significantly promoted the uptake of the native unconjugated enzymes, “equalizing” the cellular access for native vs conjugated ASNase. The screening for competing inhibitors of PTS receptor-mediated endocytosis revealed spermine and caproate/lipoate derivatives as the most potent inhibitors or antagonists, significantly reducing the cytostatic efficacy of polyamine-conjugated ASNases. The results obtained emphasize the complex, cell-type-dependent and inhibitor-specific nature of these interactions, which highlights the profound involvement of PTS in L-ASNase internalization and cytotoxic activity. These findings support the viability of polyamine conjugation as a strategy to enhance L-ASNase delivery and therapeutic efficacy by targeting the PTS. Full article