RESUMO
The paper presents data on phytosanitary monitoring of garden cenoses for fire blight in the Turkestan, Zhambyl, and Almaty regions of Kazakhstan. The purpose of this study is to assess the phytosanitary situation in various regions of Kazakhstan, determine the extent of fire blight spread, and isolate and identify the fire blight pathogen. During the study, methods such as hypersensitivity, pathogenicity, and fluorescent simplification-based specific hybridization polymerase chain reaction (FLASH-PCR) were used. It was found that in all the surveyed areas, disease foci were identified. For the first time, the fire blight pathogen was detected on fruit crops such as plum, peach, cherry plum, and quince, as well as on wild apricots. 274 plant samples were collected from which microorganisms were isolated. Isolates related to the fire blight pathogen Erwinia amylovora were identified by methods of hypersensitivity, pathogenicity, and FLASH-PCR diagnostics. Of the 156 isolates of microorganisms isolated from apple tree plant samples, 21 inhibited the in vitro growth of E. amylovora to varying degrees. Isolates 16.2 and 19.2 with maximum antagonistic activity were selected, where the pathogen growth inhibition zones were 52.2 ± 2.58 mm and 45.6 ± 0.55 mm, respectively. Based on the obtained sequence of nucleotides of the 16SpRNA gene site, it was found that the selected isolates with high antagonistic activity belonged to the Pseudomonas genus. In the future, based on these isolates, a new biological product for fire blight control can be created and adapted to the natural and climatic conditions of Kazakhstan.
Assuntos
Erwinia amylovora , Doenças das Plantas , Cazaquistão , Doenças das Plantas/microbiologia , Erwinia amylovora/isolamento & purificação , Reação em Cadeia da Polimerase , Malus/microbiologiaRESUMO
Changes in the plant microbiota composition are intimately associated with the health of the plant, but factors controlling the microbial community in flowers are poorly understood. In this study, we used apple flowers and fire blight as a model system to investigate the effects of floral microbiota and microbial competition on disease development and suppression. To compare changes in microbial flora with the RNA expression patterns of plants, the flower samples were collected in three different flowering stages (Bud, Popcorn, and Full-bloom). Using advanced sequencing technology, we analyzed the data and conducted both in vitro and in vivo experiments to validate our findings. Our results show that the Erwinia amylovora use arabinogalactan, which is secreted on the flowers, for early colonization of apple flowers. Pantoea agglomerans was more competitive for arabinogalactan than E. amylovora. Additionally, P. agglomerans suppressed the expression of virulence factors of E. amylovora by using arabinose, which is a major component of arabinogalactan, which induces virulence gene expression. The present data provide new insights into developing control strategies for diverse plant diseases, including fire blight, by highlighting the importance of nutrients in disease development or suppression.
Assuntos
Erwinia amylovora , Flores , Galactanos , Malus , Microbiota , Doenças das Plantas , Malus/microbiologia , Erwinia amylovora/patogenicidade , Erwinia amylovora/fisiologia , Doenças das Plantas/microbiologia , Flores/microbiologia , Galactanos/metabolismo , Nutrientes/metabolismo , Pantoea/fisiologia , Pantoea/genética , Pantoea/patogenicidade , Arabinose/metabolismo , Fatores de Virulência/genéticaRESUMO
FB_MR5 is a nucleotide-binding domain and leucine-rich repeat protein identified from wild apple species Malus × robusta 5 conferring disease resistance to bacterial fire blight. FB_MR5 (hereafter MrMR5) recognizes the cysteine protease effector EaAvrRpt2 secreted from the causal agent of bacterial fire blight, Erwinia amylovora. We previously reported that MrMR5 is activated by the C-terminal cleavage product (ACP3) of Malus domestica RIN4 (MdRIN4) produced by EaAvrRpt2-directed proteolysis. We show that MbMR5 from a wild apple species Malus baccata shares 99.4% amino acid sequence identity with MrMR5. Surprisingly, transient expression of MbMR5 in Nicotiana benthamiana showed autoactivity in contrast to MrMR5. Domain swap and mutational analyses revealed that 1 amino acid polymorphism in the MbMR5 CC domain is critical in enhancing autoactivity. We further demonstrated that MrMR5 carrying 7 amino acid polymorphisms present in MbMR5 is not activated by MdRIN4 ACP3 but recognizes AvrRpt2 without MdRIN4 in N. benthamiana. Our findings indicate that naturally occurring polymorphisms of MR5 natural variants can confer its cell death-inducing activity and the effector recognition mechanism likely due to altered compatibility with RIN4.
Assuntos
Resistência à Doença , Erwinia amylovora , Malus , Doenças das Plantas , Proteínas de Plantas , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Erwinia amylovora/genética , Erwinia amylovora/patogenicidade , Doenças das Plantas/microbiologia , Doenças das Plantas/genética , Doenças das Plantas/imunologia , Resistência à Doença/genética , Malus/microbiologia , Malus/genética , Malus/imunologia , Nicotiana/genética , Nicotiana/microbiologia , Nicotiana/imunologia , Nicotiana/metabolismo , Sequência de AminoácidosRESUMO
AIMS: The Gram-negative bacterium Erwinia amylovora (Ea) is the causal agent of fire blight, a devastating disease of apples and pears. In the fire blight disease cycle, Ea grows in different plant tissues, each presenting a distinct nutrient environment. Here, we investigate the ability of aspartate and tyrosine double auxotroph Ea lines to proliferate on apple flower stigma surfaces representing the epiphytic growth stage of Ea and in developing fruitlets representing one endophytic growth stage of Ea. METHODS AND RESULTS: Heterologous complementation studies in an Escherichia coli aspartate and tyrosine auxotroph verify that Ea aspartate aminotransferase (AspC) and tyrosine aminotransferase (TyrB) act as aspartate and tyrosine amino transferases. Growth analysis reveals that Ea aspC tyrB mutants multiply to near-wild-type levels on apple flower stigmas and immature fruitlets. CONCLUSIONS: Ea AspC and TyrB are reciprocally complementing for aspartate and tyrosine synthesis in Ec and in Ea. Ea aspC and tyrB mutants obtain sufficient aspartate and tyrosine to support multiplication on stigma surfaces and virulence in immature fruitlets.
Assuntos
Ácido Aspártico , Erwinia amylovora , Flores , Malus , Doenças das Plantas , Tirosina , Erwinia amylovora/genética , Erwinia amylovora/patogenicidade , Doenças das Plantas/microbiologia , Malus/microbiologia , Tirosina/metabolismo , Virulência , Ácido Aspártico/metabolismo , Flores/microbiologia , Aspartato Aminotransferases/metabolismo , Frutas/microbiologia , Tirosina Transaminase/genética , Tirosina Transaminase/metabolismoRESUMO
Erwinia amylovora, the primary causative agent of blight disease in rosaceous plants, poses a significant threat to agricultural yield worldwide, with limited effective countermeasures. The emergence of sustainable alternative agents such as bacteriophages is a promising solution for fire blight that specifically targets Erwinia. In this study, we isolated pEp_SNUABM_01 and pEa_SNUABM_55 from a South Korean apple orchard soil, analyzed their genomic DNA sequences, and performed a comprehensive comparative analysis of Hena1 in four distinct sections. This study aimed to unveil distinctive features of these phages, with a focus on host recognition, which will provide valuable insights into the evolution and characteristics of Henunavirus bacteriophages that infect plant pathogenic Erwinia spp. By elucidating the distinct genomic features of these phages, particularly in terms of host recognition, this study lays a foundation for their potential application in mitigating the risks associated with fire blight in Rosaceae plants on a global scale.
Assuntos
Bacteriófagos , Erwinia amylovora , Genoma Viral , Doenças das Plantas , Erwinia amylovora/virologia , Erwinia amylovora/genética , Doenças das Plantas/virologia , Doenças das Plantas/microbiologia , Bacteriófagos/genética , Bacteriófagos/classificação , Bacteriófagos/isolamento & purificação , Filogenia , Especificidade de Hospedeiro , Genômica , Malus/microbiologia , Malus/virologia , Microbiologia do SoloRESUMO
Bacteria, fungi, and mammals contain lactonases that can degrade the Gram-negative bacterial quorum sensing (QS) molecules N-acyl homoserine lactones (AHLs). AHLs are critical for bacteria to coordinate gene expression and pathogenicity with population density. However, AHL-degrading lactonases present variable substrate ranges, including degradation of the Pencillium expansum lactone mycotoxin patulin. We selected Erwinia spp. as our model bacteria to further investigate this interaction. We find both native apple microbiome Erwinia spp. and the fruit tree pathogen Erwinia amylovora to be inhibited by patulin. At patulin concentrations that inhibited E. amylovora growth, expression of E. amylovora lactonase encoded by EaaiiA was increased. EaAiiA demonstrated the ability to degrade patulin in vitro, as well, as in vivo where it reduced apple disease and patulin production by P. expansum. Fungal-bacterial co-cultures revealed that the E. amylovora Δeaaiia strain failed to protect apples from P. expansum infections, which contained significant amounts of patulin. Our results suggest that bacterial lactonase production can modulate the pathogenicity of P. expansum in response to the secretion of toxic patulin. IMPORTANCE: Chemical signaling in the microbial world facilitates the regulation of gene expression as a function of cell population density. This is especially true for the Gram-negative bacterial signal N-acyl homoserine lactone (AHL). Lactonases that deactivate AHLs have attracted a lot of attention because of their antibacterial potential. However, the involvement of these enzymes in inhibiting fungal pathogens and the potential role of these enzymes in bacterial-fungal interactions are unknown. Here, we find that a bacterial enzyme involved in the degradation of AHLs is also induced by and degrades the fungal lactone mycotoxin, patulin. This work supports the potential use of bacterial enzymes and/or the producing bacteria in controlling the post-harvest fruit disease caused by the patulin-producing fungus Penicillium expansum.
Assuntos
Hidrolases de Éster Carboxílico , Erwinia amylovora , Malus , Patulina , Patulina/metabolismo , Hidrolases de Éster Carboxílico/metabolismo , Hidrolases de Éster Carboxílico/genética , Malus/microbiologia , Erwinia amylovora/genética , Erwinia amylovora/efeitos dos fármacos , Erwinia amylovora/enzimologia , Erwinia amylovora/metabolismo , Doenças das Plantas/microbiologia , Penicillium/genética , Penicillium/enzimologia , Penicillium/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Interações Microbianas , Percepção de Quorum , Lactonas/metabolismo , Lactonas/farmacologiaRESUMO
Fire blight, caused by the bacterium Erwinia amylovora, is a major threat to pear production worldwide. Bacteriophages, viruses that infect bacteria, are a promising alternative to antibiotics for controlling fire blight. In this study, we isolated a novel bacteriophage, RH-42-1, from Xinjiang, China. We characterized its biological properties, including host range, plaque morphology, infection dynamics, stability, and sensitivity to various chemicals. RH-42-1 infected several E. amylovora strains but not all. It produced clear, uniform plaques and exhibited optimal infectivity at a multiplicity of infection (MOI) of 1, reaching a high titer of 9.6 × 109 plaque-forming units (PFU)/mL. The bacteriophage had a short latent period (10 min), a burst size of 207 PFU/cell, and followed a sigmoidal one-step growth curve. It was stable at temperatures up to 60 °C but declined rapidly at higher temperatures. RH-42-1 remained viable within a pH range of 5 to 9 and was sensitive to extreme pH values. The bacteriophage demonstrates sustained activity upon exposure to ultraviolet radiation for 60 min, albeit with a marginal reduction. In our assays, it exhibited a certain level of resistance to 5% chloroform (CHCl3), 5% isopropanol (C3H8O), and 3% hydrogen peroxide (H2O2), which had little effect on its activity, whereas it showed sensitivity to 75% ethanol (C2H5OH). Electron microscopy revealed that RH-42-1 has a tadpole-shaped morphology. Its genome size is 14,942 bp with a GC content of 48.19%. Based on these characteristics, RH-42-1 was identified as a member of the Tectiviridae family, Alphatectivirus genus. This is the first report of a bacteriophage in this genus with activity against E. amylovora.
Assuntos
Bacteriófagos , Erwinia amylovora , Microbiologia do Solo , Bacteriófagos/isolamento & purificação , Bacteriófagos/genética , Bacteriófagos/fisiologia , Bacteriófagos/classificação , China , Erwinia amylovora/virologia , Erwinia amylovora/efeitos dos fármacos , Genoma Viral , Especificidade de Hospedeiro , Concentração de Íons de Hidrogênio , Filogenia , Doenças das Plantas/microbiologia , Pyrus/microbiologia , Pyrus/virologiaRESUMO
Fire blight, caused by Erwinia amylovora, is a devastating bacterial disease that threatens apple and pear production. It is mainly controlled by using antibiotics, such as streptomycin. Due to development of E. amylovora resistant strains and the excessive agricultural use of antibiotics, there is an increased awareness of the possibility of antibiotic resistance gene transfer to other microbes. Urgent development of biocontrol agents (BCAs) is needed that can be incorporated into integrated pest management programs as antibiotic alternatives. A novel phage-carrier system (PCS) that combines an antagonistic bacterium, Pantoea agglomerans, with its ability to act as a phage-carrier bacterium for Erwinia phages has been developed. The low viability of P. agglomerans cells following spray-drying (SD) has been a challenge for the industrial-scale production of this PCS. Here, an SD protocol was developed for P. agglomerans by modifying the growth medium and bacterial cell formulation using D(+)-trehalose and maltodextrin. The developed protocol is amenable to the industrial-scale production of the BCA/PCS. The P. agglomerans viability was greater than 90% after SD and had a shelf life at 4 °C of 4 months, and reconstituted cells showed a 3 log reduction in E. amylovora counts with a pear disc assay.
Assuntos
Bacteriófagos , Erwinia amylovora , Malus , Pantoea , Bacteriófagos/genética , Antibacterianos/farmacologia , Erwinia amylovora/genética , Doenças das Plantas/prevenção & controle , Doenças das Plantas/microbiologiaRESUMO
Flowers are colonized by a diverse community of microorganisms that can alter plant health and interact with floral pathogens. Erwinia amylovora is a flower-inhabiting bacterium and a pathogen that infects different plant species, including Malus × domestica (apple). Previously, we showed that the co-inoculation of two bacterial strains, members of the genera Pseudomonas and Pantoea, isolated from apple flowers, reduced disease incidence caused by this floral pathogen. Here, we decipher the ecological interactions between the two flower-associated bacteria and E. amylovora in field experimentation and in vitro co-cultures. The two flower commensal strains did not competitively exclude E. amylovora from the stigma habitat, as both bacteria and the pathogen co-existed on the stigma of apple flowers and in vitro. This suggests that plant protection might be mediated by other mechanisms than competitive niche exclusion. Using a synthetic stigma exudation medium, ternary co-culture of the bacterial strains led to a substantial alteration of gene expression in both the pathogen and the two microbiota members. Importantly, the gene expression profiles for the ternary co-culture were not just additive from binary co-cultures, suggesting that some functions only emerged in multipartite co-culture. Additionally, the ternary co-culture of the strains resulted in a stronger acidification of the growth milieu than mono- or binary co-cultures, pointing to another emergent property of co-inoculation. Our study emphasizes the critical role of emergent properties mediated by inter-species interactions within the plant holobiont and their potential impact on plant health and pathogen behavior. IMPORTANCE: Fire blight, caused by Erwinia amylovora, is one of the most important plant diseases of pome fruits. Previous work largely suggested plant microbiota commensals suppressed disease by antagonizing pathogen growth. However, inter-species interactions of multiple flower commensals and their influence on pathogen activity and behavior have not been well studied. Here, we show that co-inoculating two bacterial strains that naturally colonize the apple flowers reduces disease incidence. We further demonstrate that the interactions between these two microbiota commensals and the floral pathogen led to the emergence of new gene expression patterns and a strong alteration of the external pH, factors that may modify the pathogen's behavior. Our findings emphasize the critical role of emergent properties mediated by inter-species interactions between plant microbiota and plant pathogens and their impact on plant health.
Assuntos
Erwinia amylovora , Malus , Incidência , Flores/microbiologia , Malus/genética , Malus/microbiologia , Erwinia amylovora/metabolismo , Doenças das Plantas/microbiologiaAssuntos
Arabidopsis , Erwinia amylovora , Erwinia , Malus , Arabidopsis/genética , Imunidade , Doenças das PlantasRESUMO
Erwinia amylovora, the causal agent of fire blight disease, has become a serious threat to the pome fruit industry in Korea since 2015. In this study, we showed that two new isolates of E. amylovora, Ea17-2187 and Ea19-7, obtained from pear orchards in Anseong, Korea, exhibited unique pathogenicity compared with other isolates thus far. Both were nonpathogenic to immature apple fruits but occasionally caused disease on immature pear fruits at varying reduced rates. Bioinformatic analyses revealed that their genomes are highly similar to those of the type strains TS3128 and ATCC49946 but have different mutations in essential virulence regulatory genes. Ea17-2187 has a single nucleotide substitution in rcsC, which encodes the core components of the Rcs system that activates the exopolysaccharide amylovoran production. In contrast, Ea19-7 contains a single nucleotide insertion in hrpL, which encodes a master regulator of the type III secretion system. In both cases, the mutation can cause premature termination and production of truncated gene products, disrupting virulence regulation. Introduction of the nonmutated rcsC and hrpL genes into Ea17-2187 and Ea19-7, respectively, fully recovered pathogenicity, comparable with that of TS3128; hence, these mutations were responsible for the altered pathogenicity observed. Interestingly, virulence assays on immature pear fruits showed that the hrpL mutant of Ea19-7 was still pathogenic, although its virulence level was markedly reduced. Taken together, these results suggest that the two new isolates might act as opportunistic pathogens or cheaters and that some Korean isolates might have evolved to acquire alternative pathways for activating pathogenicity factors.
Assuntos
Erwinia amylovora , Doenças das Plantas , Pyrus , Erwinia amylovora/genética , Erwinia amylovora/patogenicidade , Doenças das Plantas/microbiologia , Pyrus/microbiologia , Virulência/genética , República da Coreia , Polimorfismo de Nucleotídeo Único , Proteínas de Bactérias/genética , Malus/microbiologia , Genoma Bacteriano , Frutas/microbiologia , Polissacarídeos BacterianosRESUMO
Erwinia amylovora is a Gram-negative bacterium, responsible for the fire blight disease in Rosaceae plants. Its virulence is correlated with the production of an exopolysaccharide (EPS) called amylovoran, which protects the bacterium from the surrounding environment and helps its diffusion inside the host. Amylovoran biosynthesis relies on the expression of twelve genes clustered in the ams operon. One of these genes, amsI, encodes for a Low Molecular Weight Protein Tyrosine Phosphatase (LMW-PTP) called EaAmsI, which plays a key role in the regulation of the EPS production pathway. For this reason, EaAmsI was chosen in this work as a target for the development of new antibacterial agents against E. amylovora. To achieve this aim, a set of programs (DOCK6, OpenEye FRED) was selected to perform a virtual screening using a database of ca. 700 molecules. The six best-scoring compounds identified were tested in in vitro assays. A complete inhibition kinetic characterization carried out on the most promising molecule (n-Heptyl ß-D-glucopyranoside, N7G) showed an inhibition constant of 7.8 ± 0.6 µM. This study represents an initial step towards the development of new EaAmsI inhibitors able to act as antibacterial agents against E. amylovora infections.
Assuntos
Erwinia amylovora , Erwinia , Malus , Malus/metabolismo , Virulência , Proteínas Tirosina Fosfatases/genética , Proteínas Tirosina Fosfatases/metabolismo , Antibacterianos/farmacologia , Antibacterianos/metabolismo , Doenças das Plantas/microbiologia , Polissacarídeos Bacterianos/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Erwinia/genética , Erwinia/metabolismoRESUMO
Erwinia amylovora is a notorious plant pathogenic bacterium of global concern that has devastated the apple and pear production industry worldwide. Nevertheless, the approaches available currently to understand the genetic diversity of E. amylovora remain unsatisfactory because of the lack of a trustworthy index and data covering the globally occurring E. amylovora strains; thus, their origin and distribution pattern remains ambiguous. Therefore, there is a growing need for robust approaches for obtaining this information via the comparison of the genomic structure of Amygdaloideae-infecting strains to understand their genetic diversity and distribution. Here, the whole-genome sequences of 245 E. amylovora strains available from the NCBI database were compared to identify intraspecific genes for use as an improved index for the simple classification of E. amylovora strains regarding their distribution. Finally, we discovered two kinds of strain-typing protein-encoding genes, i.e., the SAM-dependent methyltransferase and electron transport complex subunit RsxC. Interestingly, both of these proteins carried an amino acid repeat in these strains: SAM-dependent methyltransferase comprised a single-amino-acid repeat (asparagine), whereas RsxC carried a 40-amino-acid repeat, which was differentially distributed among the strains. These noteworthy findings and approaches may enable the exploration of the genetic diversity of E. amylovora from a global perspective.
Assuntos
Erwinia amylovora , Erwinia , Malus , Rosaceae , Erwinia amylovora/genética , Erwinia amylovora/metabolismo , Aminoácidos/metabolismo , Rosaceae/microbiologia , Malus/microbiologia , Variação Genética , Metiltransferases/metabolismo , Doenças das Plantas/microbiologiaRESUMO
Several fire blight resistance loci in Malus genotypes map on different linkage groups (LGs) representing chromosomes of the domesticated apple. Prior genetics studies primarily focused on F1 populations. A strong resistance quantitative trait locus (QTL) explained up to 66% of phenotypic variance in an F1 progeny derived from crossing the highly resistant wild apple genotype Malus fusca MAL0045 and the highly susceptible apple cultivar 'Idared', which was previously mapped on LG10 (Mfu10) of MAL0045. Strains of the causative bacterial pathogen Erwinia amylovora, notably those that show a single nucleotide polymorphism in the avrRpt2EA effector protein sequence at position 156 (e.g., Ea3049), are more virulent and overcome some known fire blight resistance donors and their QTLs. However, MAL0045 is resistant to Ea3049 and Mfu10 is not overcome, but most of the F1 progeny were highly susceptible to this strain. This phenomenon led to the assumption that other putative resistance factors not segregating in the F1 progeny might be present in the genome of MAL0045. Here, we crossed F1 progeny together to obtain 135 F2 individuals. Facilitated by genotyping-by-sequencing and phenotypic assessments, we identified and mapped two novel resistance QTLs in these F2 individuals on LGs 4 and 15, which were not identified in the F1. To our knowledge, these are the first resistance QTLs mapped in F2 progeny in Malus. In addition, we report that neither MAL0045 nor Mfu10 is broken down by a highly aggressive U.S. strain, LA635, after analyses in the original F1 individuals. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.
Assuntos
Erwinia amylovora , Malus , Humanos , Locos de Características Quantitativas/genética , Malus/genética , Malus/microbiologia , Doenças das Plantas/microbiologia , Mapeamento Cromossômico , Genótipo , Erwinia amylovora/genéticaRESUMO
The YopJ group of acetylating effectors from phytopathogens of the genera Pseudomonas and Ralstonia have been widely studied to understand how they modify and suppress their host defence targets. In contrast, studies on a related group of effectors, the Eop1 group, lag far behind. Members of the Eop1 group are widely present in the Erwinia-Pantoea clade of Gram-negative bacteria, which contains phytopathogens, non-pathogens and potential biocontrol agents, implying that they may play an important role in agroecological or pathological adaptations. The lack of research in this group of YopJ effectors has left a significant knowledge gap in their functioning and role. For the first time, we perform a comparative analysis combining AlphaFold modelling, in planta transient expressions and targeted mutational analyses of the Eop1 group effectors from the Erwinia-Pantoea clade, to help elucidate their likely activity and mechanism(s). This integrated study revealed several new findings, including putative binding sites for inositol hexakisphosphate and acetyl coenzyme A and newly postulated target-binding domains, and raises questions about whether these effectors function through a catalytic triad mechanism. The results imply that some Eop1s may use a catalytic dyad acetylation mechanism that we found could be promoted by the electronegative environment around the active site.
Assuntos
Erwinia amylovora , Erwinia , Pantoea , Pseudomonas , Dickeya , Acetilcoenzima A , Doenças das Plantas/microbiologiaRESUMO
Herein, we developed a bio-functionalized solution-immersed silicon (SIS) sensor at the single-cell level to identify Erwinia amylovora (E. amylovora), a highly infectious bacterial pathogen responsible for fire blight, which is notorious for its rapid spread and destructive impact on apple and pear orchards. This method allows for ultra-sensitive measurements without pre-amplification or labeling compared to conventional methods. To detect a single cell of E. amylovora, we used Lipopolysaccharide Transporter E (LptE), which is involved in the assembly of lipopolysaccharide (LPS) at the surface of the outer membrane of E. amylovora, as a capture agent. We confirmed that LptE interacts with E. amylovora via LPS through in-house ELISA analysis, then used it to construct the sensor chip by immobilizing the capture molecule on the sensor surface modified with 3'-Aminopropyl triethoxysilane (APTES) and glutaraldehyde (GA). The LptE-based SIS sensor exhibited the sensitive and specific detection of the target bacterial cell in real time. The dose-response curve shows a linearity (R2 > 0.992) with wide dynamic ranges from 1 to 107 cells/mL for the target bacterial pathogen. The sensor showed the value change (dΨ) of approximately 0.008° for growing overlayer thickness induced from a single-cell E. amylovora, while no change in the control bacterial cell (Bacillus subtilis) was observed, or negligible change, if any. Furthermore, the bacterial sensor demonstrated a potential for the continuous detection of E. amylovora through simple surface regeneration, enabling its reusability. Taken together, our system has the potential to be applied in fields where early symptoms are not observed and where single-cell or ultra-sensitive detection is required, such as plant bacterial pathogen detection, foodborne pathogen monitoring and analysis, and pathogenic microbial diagnosis.
Assuntos
Erwinia amylovora , Lipopolissacarídeos , Bacillus subtilis , Ensaio de Imunoadsorção EnzimáticaRESUMO
The stability of the fire blight control material, oxytetracycline, in water is strongly affected by pH, increasing with increasing acidity. From 2017 to 2021, pear and apple orchard trials were conducted to evaluate if acidic amendments to oxytetracycline sprays improve fire blight control. Compared with the water-treated control, infection suppression after two bloom applications of an acidified commercial oxytetracycline formulation averaged 85.9 ± 0.4% compared with 72.2 ± 1.7% without an acidifier, but individual trials frequently had insufficient statistical power to separate among acidified and non-acidified antibiotic treatments. Across trials, a significant linear relationship was observed for regression of relative infection suppression from oxytetracycline (hydrochloride formulation) on spray tank pH. Similar relationships were observed for oxytetracycline (calcium complex formulation) and kasugamycin (P values were 0.055 and 0.069, respectively). Also based on regression, acidified oxytetracycline and kasugamycin suppressed epiphytic populations of Erwinia amylovora on flowers to a greater degree than the antibiotic only. As spray suspensions, commercial oxytetracycline formulations at label rate and amended with citric acid (1.2 g/liter) in well water had pH values near 3.4, but after spraying, the pH of flowers washed in deionized water (1 ml/flower) measured in a range of 5.2 to 5.5 compared with a pH range of 5.8 to 6.0 after a treatment of oxytetracycline only. In pear fruit finish trials, sprays acidified with citric acid-based materials had negligible effects on fruit russeting. Based on a serological assay, the detectable residual of oxytetracycline on apple foliage was increased by co-application with citric acid compared with a non-acidified control.
Assuntos
Erwinia amylovora , Malus , Oxitetraciclina , Pyrus , Oxitetraciclina/farmacologia , Doenças das Plantas/prevenção & controle , Antibacterianos/farmacologia , Ácido Cítrico , ÁguaRESUMO
Isolates of the fire blight pathogen Erwinia amylovora with high-level resistance to oxytetracycline (minimal inhibitory concentration [MIC] > 100 µg/ml) and to streptomycin (MIC > 100 µg/ml) were recovered from four commercial pear orchards in California between 2018 and 2020. The two representative oxytetracycline- and streptomycin-resistant (OxyTcR-SmR) strains 32-10 and 33-1 were as virulent as the antibiotic susceptible strain 13-1 in causing blossom blight of pear and were recovered more than 50% of the time 7 days after co-inoculation to pear flowers with strain 13-1. In the field, inoculation of strain 32-10 to pear flowers that were pretreated with oxytetracycline at 200 µg/ml did not reduce disease compared with an untreated control. Four OxyTcR-SmR strains were subjected to draft genome sequencing to identify the genetic determinants of antibiotic resistance and their location. A 43.6-kb IncX plasmid, designated pX11-7, was detected in each of the four strains, and this plasmid encoded the tetracycline-resistance gene tetB and the streptomycin-resistance gene pair strAB within a large putatively mobile genetic element consisting of the transposon Tn10 that had inserted within the streptomycin-resistance transposon Tn6082. We also determined that pX11-7 was conjugative and was transferred at a rate that was 104 to 105 higher into an E. amylovora strain isolated in California compared with an E. amylovora strain that was isolated in Michigan. The occurrence of high levels of resistance to both oxytetracycline and streptomycin in E. amylovora strains from commercial pear orchards in California significantly limits the options for blossom blight management in these locations.