Effect of allyl-isothiocyanate on survival and antimicrobial peptide expression following oral bacterial infections in Drosophila melanogaster.

Since infections with antibiotic-resistant bacteria cause increasing problems worldwide, the identification of alternative therapies is of great importance. Plant-derived bioactives, including allyl-isothiocyanate (AITC), have received attention for their antimicrobial properties. The present study therefore investigates the impact of AITC on survival and antimicrobial peptide (AMP) levels in Drosophila melanogaster challenged with the fly pathogenic bacteria Pectobacterium carotovorum subsp. carotovorum and Leuconostoc pseudomesenteroides. AITC, a sulfur-containing compound derived from glucosinolates, exhibits antimicrobial properties and has been suggested to modulate AMP expression. By using D. melanogaster, we demonstrate that AITC treatment resulted in a concentration-dependent decrease of survival rates among female flies, particularly in the presence of the Gram-negative bacterium Pectobacterium carotovorum subsp. carotovorum, whereas AITC did not affect survival in male flies. Despite the ability of isothiocyanates to induce AMP expression in cell culture, we did not detect significant changes in AMP mRNA levels in infected flies exposed to AITC. Our findings suggest sex-specific differences in response to AITC treatment and bacterial infections, underlining the complexity of host-pathogen interactions and potential limitations of AITC as a preventive or therapeutic compound at least in D. melanogaster models of bacterial infections.

Green synthesis of metal nanoparticles and study their anti-pathogenic properties against pathogens effect on plants and animals.

According to an estimate, 30% to 40%, of global fruit are wasted, leading to post harvest losses and contributing to economic losses ranging from $10 to $100 billion worldwide. Among, all fruits the discarded portion of oranges is around 20%. A novel and value addition approach to utilize the orange peels is in nanoscience. In the present study, a synthesis approach was conducted to prepare the metallic nanoparticles (copper and silver); by utilizing food waste (Citrus plant peels) as bioactive reductants. In addition, the Citrus sinensis extracts showed the reducing activity against metallic salts copper chloride and silver nitrate to form Cu-NPs (copper nanoparticles) and Ag-NPs (Silver nanoparticles). The in vitro potential of both types of prepared nanoparticles was examined against plant pathogenic bacteria Erwinia carotovora (Pectobacterium carotovorum) and pathogens effect on human health Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus). Moreover, the in vivo antagonistic potential of both types of prepared nanoparticles was examined by their interaction with against plant (potato slices). Furthermore, additional antipathogenic (antiviral and antifungal) properties were also examined. The statistical analysis was done to explain the level of significance and antipathogenic effectiveness among synthesized Ag-NPs and Cu-NPs. The surface morphology, elemental description and size of particles were analyzed by scanning electron microscopy, transmission electron microscopy, energy-dispersive spectroscopy and zeta sizer (in addition polydispersity index and zeta potential). The justification for the preparation of particles was done by UV-Vis Spectroscopy (excitation peaks at 339 nm for copper and 415 nm for silver) and crystalline nature was observed by X-ray diffraction. Hence, the prepared particles are quite effective against soft rot pathogens in plants and can also be used effectively in some other multifunctional applications such as bioactive sport wear, surgical gowns, bioactive bandages and wrist or knee compression bandages, etc.

Orostachys malacophylla (pall.) fisch extracts alleviate intestinal inflammation in Drosophila.

ETHNOPHARMACOLOGICAL RELEVANCE: Orostachys malacophylla (Pall.) Fisch (O. malacophylla) is a succulent herbaceous plant that is the Orostachys genus of Crassulaceae family. O. malacophylla has been widely used as a traditional Chinese medicine with antioxidant, anti-inflammatory, anti-febrile, antidote, anti-Toxoplasma gondii properties. However, the biological function of alleviating intestinal inflammation and key bioactive compounds were still unknown. AIM OF THE STUDY: We used a Drosophila model to study the protective effects and bioactive compounds of O. malacophylla water extract (OMWE) and butanol extract (OMBE) on intestinal inflammation. MATERIALS AND METHODS: Drosophila intestinal inflammation was induced by oral invasion of dextran sodium sulfate (DSS) or Erwinia carotovora carotovora 15 (Ecc15). We revealed the protective effects of two extracts by determining intestinal reactive oxygen species (ROS) and antimicrobial peptide (AMP) levels and intestinal integrity, and using network pharmacology analysis to identify bioactive compounds. RESULTS: We demonstrated that both OMWE and OMBE could ameliorate the detrimental effects of DSS, including a decreased survival rate, elevated ROS levels, increased cell death, excessive proliferation of ISCs, acid-base imbalance, and disruption of intestinal integrity. Moreover, the overabundance of lipid droplets (LDs) and AMPs by Ecc15 infection is mitigated by these extracts, thereby enhancing the flies' resistance to adverse stimuli. In addition, we used widely targeted metabolomics and network pharmacology analysis to identify bioactive compounds associated with IBD healing that are present in OMWE and OMBE. CONCLUSIONS: In summary, our research indicates that OMWE and OMBE significantly mitigate intestinal inflammation and have the potential to be effective therapeutic agents for IBD in humans.

Pleiotropic effects of N-acylhomoserine lactone synthase ExpI on virulence, competition, and transmission in Pectobacterium carotovorum subsp. carotovorum Pcc21.

BACKGROUND: Pectobacterium species are necrotrophic phytopathogenic bacteria that cause soft rot disease in economically important crops. The successful infection of host plants relies on interactions among virulence factors, competition, and transmission within hosts. Pectobacteria primarily produce and secrete plant cell-wall degrading enzymes (PCWDEs) for virulence. The regulation of PCWDEs is controlled by quorum sensing (QS). Thus, the QS system is crucial for disease development in pectobacteria through PCWDEs. RESULTS: In this study, we identified a Tn-insertion mutant, M2, in the expI gene from a transposon mutant library of P. carotovorum subsp. carotovorum Pcc21 (hereafter Pcc21). The mutant exhibited reduced production and secretion of PCWDEs, impaired flagellar motility, and increased sensitivity to hydrogen peroxide, resulting in attenuated soft rot symptoms in cabbage and potato tubers. Transcriptomic analysis revealed the down-regulation of genes involved in the production and secretion in the mutant, consistent with the observed phenotype. Furthermore, the Pcc21 wild-type transiently colonized in the gut of Drosophila melanogaster within 12 h after feeding, while the mutant compromised colonization phenotype. Interestingly, Pcc21 produces a bacteriocin, carocin D, to compete with other bacteria. The mutant exhibited up-regulation of carocin D-encoding genes (caroDK) and inhibited the growth of a closely related bacterium, P. wasabiae. CONCLUSION: Our results demonstrated the significance of ExpI in the overall pathogenic lifestyle of Pcc21, including virulence, competition, and colonization in plant and insect hosts. These findings suggest that disease outcome is a result of complex interactions mediated by ExpI across multiple steps. © 2023 Society of Chemical Industry.

Trichoderma-derived emodin competes with ExpR and ExpI of Pectobacterium carotovorum subsp. carotovorum to biocontrol bacterial soft rot.

BACKGROUND: Quorum sensing inhibitors (QSIs) are an emerging control tool that inhibits the quorum sensing (QS) system of pathogenic bacteria. We aimed to screen for potential QSIs in the metabolites of Trichoderma and to explore their inhibitory mechanisms. RESULTS: We screened a strain of Trichoderma asperellum LN004, which demonstrated the ability to inhibit the color development of Chromobacterium subtsugae CV026, primarily attributed to the presence of emodin as its key QSI component. The quantitative polymerase chain reaction with reverse transcription results showed that after emodin treatment of Pectobacterium carotovorum subsp. carotovorum (Pcc), plant cell wall degrading enzyme-related synthetic genes were significantly downregulated, and the exogenous enzyme synthesis gene negative regulator (rsmA) was upregulated 3.5-fold. Docking simulations indicated that emodin could be a potential ligand for ExpI and ExpR proteins because it exhibited stronger competition than the natural ligands in Pcc. In addition, western blotting showed that emodin attenuated the degradation of n-acylhomoserine lactone on the ExpR protein and protected it. Different concentrations of emodin reduced the activity of pectinase, cellulase, and protease in Pcc by 20.81%-72.21%, 8.38%-52.73%, and 3.57%-47.50%. Lesion size in Chinese cabbages, carrots and cherry tomatoes following Pcc infestation was reduced by 10.02%-68.57%, 40.17%-88.56% and 11.36%-86.17%. CONCLUSION: Emodin from T. asperellum LN004 as a QSI can compete to bind both ExpI and ExpR proteins, interfering with the QS of Pcc and reducing the production of virulence factors. The first molecular mechanism reveals the ability of emodin as a QSI to competitively inhibit two QS proteins simultaneously. © 2023 Society of Chemical Industry.

Transcriptional Response of Pectobacterium carotovorum to Cinnamaldehyde Treatment.

Cinnamaldehyde is a natural compound extracted from cinnamon bark essential oil, acclaimed for its versatile properties in both pharmaceutical and agricultural fields, including antimicrobial, antioxidant, and anticancer activities. Although potential of cinnamaldehyde against plant pathogenic bacteria like Agrobacterium tumefaciens and Pseudomonas syringae pv. actinidiae causative agents of crown gall and bacterial canker diseases, respectively has been documented, indepth studies into cinnamaldehyde's broader influence on plant pathogenic bacteria are relatively unexplored. Particularly, Pectobacterium spp., gram-negative soil-borne pathogens, notoriously cause soft rot damage across a spectrum of plant families, emphasizing the urgency for effective treatments. Our investigation established that the Minimum Inhibitory Concentrations (MICs) of cinnamaldehyde against strains P. odoriferum JK2, P. carotovorum BP201601, and P. versatile MYP201603 were 250 µg/ml, 125 µg/ml, and 125 µg/ml, respectively. Concurrently, their Minimum Bactericidal Concentrations (MBCs) were found to be 500 µg/ml, 250 µg/ml, and 500 µg/ml, respectively. Using RNA-sequencing analysis, we identified 1,907 differentially expressed genes in P. carotovorum BP201601 treated with 500 µg/ml cinnamaldehyde. Notably, our results indicate that cinnamaldehyde upregulated nitrate reductase pathways while downregulating the citrate cycle, suggesting a potential disruption in the aerobic respiration system of P. carotovorum during cinnamaldehyde exposure. This study serves as a pioneering exploration of the transcriptional response of P. carotovorum to cinnamaldehyde, providing insights into the bactericidal mechanisms employed by cinnamaldehyde against this bacterium.

Paraburkholderia sabiae Uses One Type VI Secretion System (T6SS-1) as a Powerful Weapon against Notorious Plant Pathogens.

Paraburkholderia sabiae LMG24235 is a nitrogen-fixing betaproteobacterium originally isolated from a root nodule of Mimosa caesalpiniifolia in Brazil. We show here that this strain effectively kills strains from several bacterial families (Burkholderiaceae, Pseudomonadaceae, Enterobacteriaceae) which include important plant pathogens in a contact-dependent manner. De novo assembly of the first complete genome of P. sabiae using long sequencing reads and subsequent annotation revealed two gene clusters predicted to encode type VI secretion systems (T6SS), which we named T6SS-1 and T6SS-3 according to previous classification methods (G. Shalom, J. G. Shaw, and M. S. Thomas, Microbiology, 153:2689-2699, 2007, https://doi.org/10.1099/mic.0.2007/006585-0). We created P. sabiae with mutations in each of the two T6SS gene clusters that abrogated their function, and the T6SS-1 mutant was no longer able to outcompete other strains in a contact-dependent manner. Notably, our analysis revealed that T6SS-1 is essential for competition against several important plant pathogens in vitro, including Burkholderia plantarii, Ralstonia solanacearum, Pseudomonas syringae, and Pectobacterium carotovorum. The 9-log reduction in P. syringae cells in the presence of P. sabiae was particularly remarkable. Importantly, in an in vivo assay, P. sabiae was able to protect potato tubers from bacterial soft rot disease caused by P. carotovorum, and this protection was partly dependent on T6SS-1. IMPORTANCE Rhizobia often display additional beneficial traits such as the production of plant hormones and the acquisition of limited essential nutrients that improve plant growth and enhance plant yields. Here, we show that the rhizobial strain P. sabiae antagonizes important phytopathogens such as P. carotovorum, P. syringae, and R. solanacearum and that this effect is due to contact-dependent killing mediated by one of two T6SS systems identified in the complete, de novo assembled genome sequence of P. sabiae. Importantly, co-inoculation of Solanum tuberosum tubers with P. sabiae also resulted in a drastic reduction of soft rot caused by P. carotovorum in an in vivo model system. This result highlights the protective potential of P. sabiae against important bacterial plant diseases, which makes it a valuable candidate for application as a biocontrol agent. It also emphasizes the particular potential of rhizobial inoculants that combine several beneficial effects such as plant growth promotion and biocontrol for sustainable agriculture.

Unleashing the Influence of cAMP Receptor Protein: The Master Switch of Bacteriocin Export in Pectobacterium carotovorum subsp. carotovorum.

Pectobacterium carotovorum subsp. carotovorum (Pcc) is a Gram-negative phytopathogenic bacterium that produces carocin, a low-molecular-weight bacteriocin that can kill related strains in response to factors in the environment such as UV exposure or nutritional deficiency. The function of the catabolite activator protein (CAP), also known as the cyclic AMP receptor protein (CRP), as a regulator of carocin synthesis was examined. The crp gene was knocked out as part of the investigation, and the outcomes were assessed both in vivo and in vitro. Analysis of the DNA sequence upstream of the translation initiation site of carocin S3 revealed two putative binding sites for CRP that were confirmed using a biotinylated probe pull-down experiment. This study revealed that the deletion of crp inhibited genes involved in extracellular bacteriocin export via the flagellar type III secretion system and impacted the production of many low-molecular-weight bacteriocins. The biotinylated probe pull-down test demonstrated that when UV induction was missing, CRP preferentially attached to one of the two CAP sites while binding to both when UV induction was present. In conclusion, our research aimed to simulate the signal transduction system that controls the expression of the carocin gene in response to UV induction.

A Novel Based Synthesis of Silver/Silver Chloride Nanoparticles from Stachys emodi Efficiently Controls Erwinia carotovora, the Causal Agent of Blackleg and Soft Rot of Potato.

In recent years, the biological synthesis of silver nanoparticles has captured researchers' attention due to their unique chemical, physical and biological properties. In this study, we report an efficient, nonhazardous, and eco-friendly method for the production of antibacterial silver/silver chloride nanoparticles utilizing the leaf extract of Stachys emodi. The synthesis of se-Ag/AgClNPs was confirmed using UV-visible spectroscopy, DPPH free radical scavenging activity, Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM), and X-ray diffraction (XRD). An intense peak absorbance was observed at 437 nm from the UV-visible analysis. The Stachys emodi extract showed the highest DPPH scavenging activity (89.4%). FTIR analysis detected various bands that indicated the presence of important functional groups. The SEM morphological study revealed spherical-shaped nanoparticles having a size ranging from 20 to 70 nm. The XRD pattern showed the formation of a spherical crystal of NPs. The antibacterial activity performed against Erwinia carotovora showed the maximum inhibition by centrifuged silver nanoparticles alone (se-Ag/AgClNPs) and in combination with leaf extract (se-Ag/AgClNPs + LE) and leaf extract (LE) of 98%, 93%, and 62% respectively. These findings suggested that biosynthesized NPs can be used to control plant pathogens effectively.

Occurrence, Characteristics, and qPCR-Based Identification of Pectobacterium versatile Causing Soft Rot of Chinese Cabbage in China.

Pectobacterium is one of the most important genera of phytopathogenic bacteria. It can cause soft-rot diseases on a wide range of plant species across the world. In this study, three Pectobacterium strains (KC01, KC02, and KC03) were isolated from soft-rotted Chinese cabbage in Beijing, China. These three strains were identified as Pectobacterium versatile based on phylogenetic analysis of Pectobacterium 16S ribosomal RNA, pmrA, and 504 Pectobacterium core genes, as well as a genomic average nucleotide identity analysis. Their biochemical characteristics were found to be similar to the P. versatile type strain ICMP9168T but differed in response to citric acid, stachyose, D-glucuronic acid, dextrin, and N-acetyl-ß-D-mannosamine. All of the tested P. versatile strains showed different carbohydrate utilization abilities compared with P. carotovorum and P. odoriferum, particularly in their ability to utilize D-arabitol, L-rhamnose, and L-serine. Under laboratory conditions, the maceration ability of P. versatile on Chinese cabbage was the highest at 28°C, compared with those at 13, 28, 23, and 33°C. Additionally, P. versatile could infect all of the 17 known Pectobacterium host plants, except for Welsh onion (Allium fistulosum). A SYBR Green quantitative PCR (qPCR) detection system was developed to distinguish P. versatile from other soft-rot bacteria based on the combined performance of melting curve (with a single melting peak at around 85°C) and fluorescence curve (with cycle threshold <30) when the bacterial genomic DNA concentration was in the range of 10 pg/µl to 10 ng/µl. This study is the first to report the presence of P. versatile on Chinese cabbage in China, as well as a specific and sensitive qPCR assay that can be used to quickly identify P. versatile. The work contributes to a better understanding of P. versatile and will facilitate the effective diagnosis of soft-rot disease, ultimately benefitting commercial crop production.

Innovative biosynthesis, artificial intelligence-based optimization, and characterization of chitosan nanoparticles by Streptomyces microflavus and their inhibitory potential against Pectobacterium carotovorum.

Microbial-based strategy in nanotechnology offers economic, eco-friendly, and biosafety advantages over traditional chemical and physical protocols. The current study describes a novel biosynthesis protocol for chitosan nanoparticles (CNPs), employing a pioneer Streptomyces sp. strain NEAE-83, which exhibited a significant potential for CNPs biosynthesis. It was identified as Streptomyces microflavus strain NEAE-83 based on morphological, and physiological properties as well as the 16S rRNA sequence (GenBank accession number: MG384964). CNPs were characterized by SEM, TEM, EDXS, zeta potential, FTIR, XRD, TGA, and DSC. CNPs biosynthesis was maximized using a mathematical model, face-centered central composite design (CCFCD). The highest yield of CNPs (9.41 mg/mL) was obtained in run no. 27, using an initial pH of 5.5, 1% chitosan, 40 °C, and a 12 h incubation period. Innovatively, the artificial neural network (ANN), was used for validating and predicting CNPs biosynthesis based on the trials data of CCFCD. Despite the high precision degree of both models, ANN was supreme in the prediction of CNPs biosynthesis compared to CCFCD. ANN had a higher prediction efficacy and, lower error values (RMSE, MDA, and SSE). CNPs biosynthesized by Streptomyces microflavus strain NEAE-83 showed in-vitro antibacterial activity against Pectobacterium carotovorum, which causes the potato soft rot. These results suggested its potential application for controlling the destructive potato soft rot diseases. This is the first report on the biosynthesis of CNPs using a newly isolated; Streptomyces microflavus strain NEAE-83 as an eco-friendly approach and optimization of the biosynthesis process by artificial intelligence.

Effects of Rhapontigenin as a Novel Quorum-Sensing Inhibitor on Exoenzymes and Biofilm Formation of Pectobacterium carotovorum subsp. carotovorum and Its Application in Vegetables.

The aim of this study was to devise a method to protect Chinese cabbage (Brassica chinensis) and lettuce (Lactuca sativa) from bacterial-disease-induced damage during storage. Thus, the potential of rhapontigenin as a quorum sensing (QS) inhibitor against Pectobacterium carotovorum subsp. carotovorum (P. carotovorum) was evaluated. The QS inhibitory effects of rhapontigenin were confirmed by significant inhibition of the production of violacein in Chromobacterium violaceum CV026 (C. violaceum, CV026). The inhibitory effects of rhapontigenin on the motility, exopolysaccharide (EPS) production, biofilm formation and virulence−exoenzyme synthesis of P. carotovorum were investigated. Acyl-homoserine lactones (AHLs) were quantified using liquid chromatography−mass spectrometry (LC−MS). The inhibitory effects of rhapontigenin on the development of biofilms were observed using fluorescence microscopy and scanning electron microscopy (SEM). A direct-inoculation assay was performed to investigate the QS inhibitory effects of rhapontigenin on P. carotovorum in Chinese cabbage and lettuce. Our results demonstrated that rhapontigenin exhibited significant inhibition (p < 0.05) of the motility, EPS production, biofilm formation, virulence−exoenzyme synthesis and AHL production of P. carotovorum. Additionally, the result of the direct-inoculation assay revealed that rhapontigenin might provide vegetables with significant shelf-life extension and prevent quality loss by controlling the spread of soft-rot symptoms. Consequently, the study provided a significant insight into the potential of rhapontigenin as a QS inhibitor against P. carotovorum.

Pectobacterium carotovorum Phage vB_PcaM_P7_Pc Is a New Member of the Genus Certrevirus.

Pectobacterium carotovorum is an economically important phytopathogen and has been identified as the major causative agent of bacterial soft rot in carrots. Control of this phytopathogen is vital to minimizing carrot harvest losses. As fully efficient control measures to successfully avoid the disease are unavailable, the phage-mediated biocontrol of the pathogen has recently gained scientific attention. In this study, we present a comprehensive characterization of the P. carotovorum phage vB_PcaM_P7_Pc (abbreviated as P7_Pc) that was isolated from infected carrot samples with characteristic soft rot symptoms, which were obtained from storage facilities at market places in Gampaha District, Sri Lanka. P7_Pc is a myovirus, and it exhibits growth characteristics of an exclusively lytic life cycle. It showed visible lysis against four of the tested P. carotovorum strains and one Pectobacterium aroidearum strain. This phage also showed a longer latent period (125 min) than other related phages; however, this did not affect its high phage titter (>1010 PFU/mL). The final assembled genome of P7_Pc is 147,299 bp in length with a G+C content of 50.34%. Of the 298 predicted open reading frames (ORFs) of the genome of P7_Pc, putative functions were assigned to 53 ORFs. Seven tRNA-coding genes were predicted in the genome, while the genome lacked any major genes coding for lysogeny-related products, confirming its virulent nature. The P7_Pc genome shares 96.12% and 95.74% average nucleotide identities with Cronobacter phages CR8 and PBES02, respectively. Phylogenetic and phylogenomic analyses of the genome revealed that P7_Pc clusters well within the clade with the members representing the genus Certrevirus. Currently, there are only 4 characterized Pectobacterium phages (P. atrosepticum phages phiTE and CB7 and Pectobacterium phages DU_PP_I and DU_PP_IV) that are classified under the genus, making the phage P7_Pc the first reported member of the genus isolated using the host bacterium P. carotovorum. The results of this study provide a detailed characterization of the phage P7_Pc, enabling its careful classification into the genus Certrevirus. The knowledge gathered on the phage based on the shared biology of the genus will further aid in the future selection of phage P7_Pc as a biocontrol agent. IMPORTANCE Bacterial soft rot disease, caused by Pectobacterium spp., can lead to significant losses in carrot yields. As current control measures involving the use of chemicals or antibiotics are not recommended in many countries, bacteriophage-mediated biocontrol strategies are being explored for the successful control of these phytopathogens. The successful implementation of such biocontrol strategies relies heavily upon the proper understanding of the growth characteristics and genomic properties of the phage. Further, the selection of taxonomically different phages for the formulation of phage cocktails in biocontrol applications is critical to combat potential bacterial resistance development. This study was conducted to carefully characterize and resolve the phylogenetic placement of the P. carotovorum phage vB_PcaM_P7_Pc by using its biological and genomic properties. Phage P7_Pc has a myovirus morphotype with an exclusively lytic life cycle, and the absence of genes related to lysogeny, toxin production, and antibiotic resistance in its genome confirmed its suitability to be used in environmental applications. Furthermore, P7_Pc is classified under the genus Certrevirus, making it the first reported phage of the genus of the host species, P. carotovorum.

Control of the bacterial soft rot pathogen, Pectobacterium carotovorum by Bacillus velezensis CE 100 in cucumber.

Pectobacterium carotovorum is a problematic bacterial pathogen causing soft rot in different vegetable crops, resulting in yield losses during pre- and post-harvest periods. In this study, Bacillus velezensis CE 100 showed antibacterial activity against P. carotovorum. Co-inoculation experiment indicated that B. velezensis CE 100 reduced the proliferation rate of P. carotovorum at the early incubation period and that a long incubation time induced a loss of viability of the bacterial pathogen. Agar well diffusion assay revealed that the culture filtrate of strain CE 100 affected the growth of P. carotovorum in a dose-dependent pattern. In time-kill assay, inoculation of P. carotovorum with 50% culture filtrate of strain CE 100 resulted in a complete loss of survival at 4 h incubation period. An antibacterial compound isolated from chloroform extract of B. velezensis CE 100 was identified as macrolactin A based on results of 1H and 13C NMR and mass spectrometry. However, time-kill assay showed that purified macrolactin A at a concentration of 200 µgmL-1 was not highly effective to control the growth of P. carotovorum although reduction in cell number of P. carotovorum was observed. Moreover, in vivo assay revealed that B. velezensis CE 100 effectively controlled bacterial soft rot. As a consequence, it significantly improved cucumber growth. Therefore, B. velezensis CE 100 could be used as an eco-friendly bioagent for effective control of bacterial soft rot to minimize global economic losses in crop production.

Protease Inhibitors from Solanum chacoense Inhibit Pectobacterium Virulence by Reducing Bacterial Protease Activity and Motility.

Potato is a major staple crop, and necrotrophic bacterial pathogens such as Pectobacterium spp. are a major threat to global food security. Most lines of cultivated potato (Solanum tuberosum) are susceptible to Pectobacterium spp., but some lines of wild potato are resistant, including Solanum chacoense M6. Despite the discovery of resistance in wild potatoes, specific resistance genes are yet to be discovered. Crude protein extract from M6 had a global effect on Pectobacterium brasiliense Pb1692 (Pb1692) virulence phenotypes. Specifically, M6 protein extracts resulted in reduced Pectobacterium exo-protease activity and motility, induced cell elongation, and affected bacterial virulence and metabolic gene expression. These effects were not observed from protein extracts of susceptible potato S. tuberosum DM1. A proteomics approach identified protease inhibitors (PIs) as candidates for S. chacoense resistance, and genomic analysis showed higher abundance and diversity of PIs in M6 than in DM1. We cloned five PIs that are unique or had high abundance in M6 compared with DM1 and purified the proteins (g18987, g28531, g39249, g40384, g6571). Four of the PIs significantly reduced bacterial protease activity, with strongest effects from g28531 and g6571. Three PIs (g18987, g28531, g6571) inhibited disease when co-inoculated with Pectobacterium pathogens into potato tubers. Two PIs (g28531, g6571) also significantly reduced Pb1692 motility and are promising as resistance genes. These results show that S. chacoense PIs contribute to bacterial disease resistance by inhibiting exo-proteases, motility, and tuber maceration and by modulating cell morphology and metabolism. [Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Development of a Bacteriophage Cocktail against Pectobacterium carotovorum Subsp. carotovorum and Its Effects on Pectobacterium Virulence.

Pectobacterium carotovorum subsp. carotovorum is a necrotrophic plant pathogen that secretes plant cell wall-degrading enzymes (PCWDEs) that cause soft rot disease in various crops. Bacteriophages have been under consideration as harmless antibacterial agents to replace antibiotics and copper-based pesticides. However, the emergence of bacteriophage resistance is one of the main concerns that should be resolved for practical phage applications. In this study, we developed a phage cocktail with three lytic phages that recognize colanic acid (phage POP12) or flagella (phages POP15 and POP17) as phage receptors to minimize phage resistance. The phage cocktail effectively suppressed the emergence of phage-resistant P. carotovorum subsp. carotovorum compared with single phages in in vitro challenge assays. The application of the phage cocktail to napa cabbage (Brassica rapa subsp. pekinensis) resulted in significant growth retardation of P. carotovorum subsp. carotovorum (P < 0.05) and prevented the symptoms of soft rot disease. Furthermore, phage cocktail treatments of young napa cabbage leaves in a greenhouse environment indicated effective prevention of soft rot disease compared to that in the nonphage negative control. We isolated 15 phage-resistant mutants after a phage cocktail treatment to assess the virulence-associated phenotypes compared to those of wild-type (WT) strain Pcc27. All mutants showed reduced production of four different PCWDEs, leading to lower levels of tissue softening. Ten of the 15 phage-resistant mutants additionally exhibited decreased swimming motility. Taken together, these results show that the phage cocktail developed here, which targets two different types of phage receptors, provides an effective strategy for controlling P. carotovorum subsp. carotovorum in agricultural products, with a potential ability to attenuate P. carotovorum subsp. carotovorum virulence. IMPORTANCE Pectobacterium carotovorum subsp. carotovorum is a phytopathogen that causes soft rot disease in various crops by producing plant cell wall-degrading enzymes (PCWDEs). Although antibiotics and copper-based pesticides have been extensively applied to inhibit P. carotovorum subsp. carotovorum, the emergence of antibiotic-resistant bacteria and demand for harmless antimicrobial products have emphasized the necessity of finding alternative therapeutic strategies. To address this problem, we developed a phage cocktail consisting of three P. carotovorum subsp. carotovorum-specific phages that recognize colanic acids and flagella of P. carotovorum subsp. carotovorum. The phage cocktail treatments significantly decreased P. carotovorum subsp. carotovorum populations, as well as soft rot symptoms in napa cabbage. Simultaneously, they resulted in virulence attenuation in phage-resistant P. carotovorum subsp. carotovorum, which was represented by decreased PCWDE production and decreased flagellum-mediated swimming motility. These results suggested that preparations of phage cocktails targeting multiple receptors would be an effective approach to biocontrol of P. carotovorum subsp. carotovorum in crops.

Effects of Natural Rheum tanguticum on the Cell Wall Integrity of Resistant Phytopathogenic Pectobacterium carotovorum subsp. Carotovorum.

The abuse of agricultural antibiotics has led to the emergence of drug-resistant phytopathogens. Rifampicin and streptomycin and streptomycin resistance Pectobacterium carotovorum subsp. carotovorum (PccS1) was obtained from pathological plants in a previous experiment. Rheum tanguticum, derived from the Chinese plateau area, exhibits excellent antibacterial activity against PccS1, yet the action mode has not been fully understood. In present text, the cell wall integrity of the PccS1 was tested by the variation of the cellular proteins, SDS polyacrylamide gel electrophoresis (SDS-PAGE), scanning electron microscopy (SEM) and Fourier transform infrared spectrophotometer (FTIR) characteristics. Label-free quantitative proteomics was further used to identify the DEPs in the pathogen response to treatment with Rheum tanguticum Maxim. ex Balf. extract (abbreviated as RTMBE). Based on the bioinformatics analysis of these different expressed proteins (DEPs), RTMBE mainly inhibited some key protein expressions of beta-Lactam resistance, a two-component system and phosphotransferase system. Most of these membrane proteins were extraordinarily suppressed, which was also consistent with the morphological tests. In addition, from the downregulated flagellar motility related proteins, it was also speculated that RTMBE played an essential antibacterial role by affecting the swimming motility of the cells. The results indicated that Rheum tanguticum can be used to attenuate the virulence of the drug-resistant phytopathogenic bacteria.

Oak bark (Quercus sp. cortex) protects plants through the inhibition of quorum sensing mediated virulence of Pectobacterium carotovorum.

Bacterial intercellular communication mediated by small diffusible molecules, known as quorum sensing (QS), is a common mechanism for regulating bacterial colonisation strategies and survival. Influence on QS by plant-derived molecules is proposed as a strategy for combating phytopathogens by modulating their virulence. This work builds upon other studies that have revealed plant-derived QS inhibitors extracted from oak bark (Quercus sp.). It was found that co-incubation of Pectobacterium carotovorum VKM-B-1247 with oak bark extract (OBE) reduced the production of acyl-HSL. This was accompanied by a dose-dependent decrease in the bacterial cellulolytic and protease activity. At the transcriptomic level, the OBE treatment suppressed the main QS-related genes expR/expI. Potato tubers pre-treated with OBE showed resistance to a manifestation of soft-rot symptoms. Analysis of the component composition of the OBE identified several biologically active molecules, such as n-hexadecanoic acid, 2,6-di-tert-butyl-4-methylphenol, butylated hydroxytoluene (BHT), gamma-sitosterol, lupeol, and others. Molecular docking of the binding energy between identified molecules and homology models of LuxR-LuxI type proteins allow to identify potential inhibitors. Collectively, obtained results figure out great potential of widely distributed oak-derived plant material for bacterial control during storage of potato.

Volatile Organic Compounds and Physiological Parameters as Markers of Potato (Solanum tuberosum L.) Infection with Phytopathogens.

The feasibility of early disease detection in potato seeds storage monitoring of volatile organic compounds (VOCs) and plant physiological markers was evaluated using 10 fungal and bacterial pathogens of potato in laboratory-scale experiments. Data analysis of HS-SPME-GC-MS revealed 130 compounds released from infected potatoes, including sesquiterpenes, dimethyl disulfide, 1,2,4-trimethylbenzene, 2,6,11-trimethyldodecane, benzothiazole, 3-octanol, and 2-butanol, which may have been associated with the activity of Fusarium sambucinum, Alternaria tenuissima and Pectobacterium carotovorum. In turn, acetic acid was detected in all infected samples. The criteria of selection for volatiles for possible use as incipient disease indicators were discussed in terms of potato physiology. The established physiological markers proved to demonstrate a negative effect of phytopathogens infecting seed potatoes not only on the kinetics of stem and root growth and the development of the entire root system, but also on gas exchange, chlorophyll content in leaves, and yield. The negative effect of phytopathogens on plant growth was dependent on the time of planting after infection. The research also showed different usefulness of VOCs and physiological markers as the indicators of the toxic effect of inoculated phytopathogens at different stages of plant development and their individual organs.

Removal of bacterial plant pathogens in columns filled with quartz and natural sediments under anoxic and oxygenated conditions.

Irrigation with surface water carrying plant pathogens poses a risk for agriculture. Managed aquifer recharge enhances fresh water availability while simultaneously it may reduce the risk of plant diseases by removal of pathogens during aquifer passage. We compared the transport of three plant pathogenic bacteria with Escherichia coli WR1 as reference strain in saturated laboratory column experiments filled with quartz sand, or sandy aquifer sediments. E. coli showed the highest removal, followed by Pectobacterium carotovorum, Dickeya solani and Ralstonia solanacearum. Bacterial and non-reactive tracer breakthrough curves were fitted with Hydrus-1D and compared with colloid filtration theory (CFT). Bacterial attachment to fine and medium aquifer sand under anoxic conditions was highest with attachment rates of max. katt1 = 765 day-1 and 355 day-1, respectively. Attachment was the least to quartz sand under oxic conditions (katt1 = 61 day-1). In CFT, sticking efficiencies were higher in aquifer than in quartz sand but there was no differentiation between fine and medium aquifer sand. Overall removal ranged between < 6.8 log10 m-1 in quartz and up to 40 log10 m-1 in fine aquifer sand. Oxygenation of the anoxic aquifer sediments for two weeks with oxic influent water decreased the removal. The results highlight the potential of natural sand filtration to sufficiently remove plant pathogenic bacteria during aquifer storage.