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.

Multilocus sequence and phenotypic analysis of Pectobacterium and Dickeya type strains for identification of soft rot Pectobacteriaceae from symptomatic potato stems and tubers in Pennsylvania.

Outbreaks of potato blackleg and soft rot caused by Pectobacterium species and more recently Dickeya species across the U.S. mid-Atlantic region have caused yield loss due to poor emergence as well as losses from stem and tuber rot. To develop management strategies for soft rot diseases, we must first identify which members of the soft rot Pectobacteriaceae are present in regional potato plantings. However, the rapidly expanding number of soft rot Pectobacteriaceae species and the lack of readily available comparative data for type strains of Pectobacterium and Dickeya hinder quick identification. This manuscript provides a comparative analysis of soft rot Pectobacteriaceae and a comprehensive comparison of type strains from this group using rep-PCR, MLSA and 16S sequence analysis, as well as phenotypic and physiological analyses using Biolog GEN III plates. These data were used to identify isolates cultured from symptomatic potato stems collected between 2016 and 2018. The isolates were characterized for phenotypic traits and by sequence analysis to identify the bacteria from potatoes with blackleg and soft rot symptoms in Pennsylvania potato fields. In this survey, P. actinidiae, P. brasiliense, P. polonicum, P. polaris, P. punjabense, P. parmentieri, and P. versatile were identified from Pennsylvania for the first time. Importantly, the presence of P. actinidiae in Pennsylvania represents the first report of this organism in the U.S. As expected, P. carotorvorum and D. dianthicola were also isolated. In addition to a resource for future work studying the Dickeya and Pectobacterium associated with potato blackleg and soft rot, we provide recommendations for future surveys to monitor for quarantine or emerging soft rot Pectobacteriace regionally.

Pseudomonas fluorescens MFE01 delivers a putative type VI secretion amidase that confers biocontrol against the soft-rot pathogen Pectobacterium atrosepticum.

The type VI secretion system (T6SS) is a contractile nanomachine widespread in Gram-negative bacteria. The T6SS injects effectors into target cells including eukaryotic hosts and competitor microbial cells and thus participates in pathogenesis and intermicrobial competition. Pseudomonas fluorescens MFE01 possesses a single T6SS gene cluster that confers biocontrol properties by protecting potato tubers against the phytopathogen Pectobacterium atrosepticum (Pca). Here, we demonstrate that a functional T6SS is essential to protect potato tuber by reducing the pectobacteria population. Fluorescence microscopy experiments showed that MFE01 displays an aggressive behaviour with an offensive T6SS characterized by continuous and intense T6SS firing activity. Interestingly, we observed that T6SS firing is correlated with rounding of Pectobacterium cells, suggesting delivery of a potent cell wall targeting effector. Mutagenesis coupled with functional assays then revealed that a putative T6SS secreted amidase, Tae3Pf , is mainly responsible for MFE01 toxicity towards Pca. Further studies finally demonstrated that Tae3Pf is toxic when produced in the periplasm, and that its toxicity is counteracted by the Tai3Pf inner membrane immunity protein.

Nano-metals forming bacteria in Egypt. II. Efficacy towards biomolecules, ultrastructure, growth parameters, and eco-friendly therapeutic of soft rot/blackleg genera.

The nanoparticles (NPs) formed by Enterococcus thailandicus, Pseudomonas putida, Marinobacter hydrocarbonoclasticus, and P. geniculate were tested against soft rot/blackleg genera. The effects of NPs recorded on bacterial DNA, proteins, and carbohydrates concentration of Pectobacterium carotovorum subsp. carotovorum, Enterobacter cloacae (soft rot), and Dickeya solani (soft rot/blackleg). Treated cells showed degradation in isolated DNA, decreased proteins and carbohydrates concentration compared with untreated cells. Using Scanning Electron Microscope (SEM), the treated cells showed collapsed and small pits in the cell wall. Using Transmission Electron Microscope (TEM), internal changes showed penetration of NPs inside the tested bacterial cells, the appearance of periplasmic space, formation of vacuoles, and condensation of cytoplasm. Disease severity ex vivo of potato tuber infected with tested genera demonstrated that NPs treatment didn't show any rotted tissue compared with untreated. The ability to uptake and accumulate FeNPs from the soil in potato (Solanum tuberosum) seedlings; Inductively Coupled Plasma-Optical Emission Spectroscopy (ICP-OES) was used. It recorded an increase in iron content of treated potato (Solanum tuberosum) seedlings with NPs, compared with untreated. FeNPs can be used to control soft rot/blackleg diseases, instead of copper pesticides. It could be a new, approach for disease management and increase the plant's nutritional value.

Genome Sequence Resource of Pectobacterium polaris QK413-1 that Causes Blackleg on Potato in Fujian Province, China.

The Pectobacterium pathogens cause soft rot and blackleg diseases on many plants and crops, including potatoes. Here, we first report a high-quality genome assembly and announcement of the P. polaris strain QK413-1, which causes blackleg disease in potatoes in China. The QK413-1 genome was sequenced and assembled using the PacBio Sequel II and Illumina sequencing platform. The assembled genome has a total size of 5,005,507 bp with a GC content of 51.81%, encoding 4,782 open reading frames, including 639 virulence genes, 273 drug resistance genes, and 416 secreted proteins. The QK413-1 genome sequence provides a valuable resource for the control of potato blackleg and research into its mechanism.

Biological control of soft rot in potato by κ-carrageenan carriers encapsulated microbial predators.

The Pectobacterium and Dickeya pectinolytic bacteria are phytopathogens responsible for several macerating diseases on a wide range of crops and ornamental plants. Recently, bacterial predators belonging to the Bdellovibrio and like organisms (BALOs) were shown to efficiently prey on these rot-causing bacteria and reduce soft rot-induced potato slice maceration. In the current research, our novel approach aimed at developing and studying a κ-carrageenan-based encapsulation system for fast-release of entrapped B. bacteriovorus HD100 in high numbers to prevent bacterial soft-rot infections. κ-carrageenan-dried carriers swelled and dissolved upon immersion in water due to a loss of potassium ions which are the main cross-linking agents. Survival rates of the predators after drying were higher for immobilized bdelloplasts (e.g., predator inside the host) compared to attack phase (host-searching, AP) cells, and with the addition of the osmoprotectant trehalose to the carriers. Released encapsulated predators preyed efficiently on soft rot bacteria, with bdelloplasts performing better as compared to AP cells. However, predation dynamics were influenced by the type of added osmoprotectant. Carrageenan-trehalose carriers encapsulating predators were able to reduce soft-rot disease in situ using a potato slice assay. To our knowledge, this research is the first to explore the potential of encapsulated BALOs against phytopathogens. KEY POINTS: • Dissolution of the carriers was affected by potassium concentration in the system. • Encapsulation of bdelloplasts with trehalose best maintained the predator viability. • The encapsulated predators efficiently controlled soft rot in vitro and in situ.

The structure of an abequose - containing O-polysaccharide isolated from Pectobacterium aquaticum IFB5637.

Soft rot and blackleg diseases, caused by pectinolytic bacteria from the numerous species of Dickeya and Pectobacterium, pose a serious threat to the world potato production. Besides, infections triggered by these pectinolytic bacteria lead to huge economic losses in the cultivation of other crops, vegetables, and ornamentals. Strains belonging to the genus Pectobacterium tend to be isolated from various environments such as rotten or asymptomatic plants, weeds, soil or water. The main virulence factors of these phytopathogenic bacteria involve plant cell wall degrading enzymes (PCWDEs) i.e. pectinases, cellulases and proteases. Among accessory virulence factors, there is often lipopolysaccharide (LPS) listed. This constituent of the external part of bacterial cell wall contains lipid A, inner and outer core in addition to O-polysaccharide (OPS). LPS plays an important role in plant-microbe interactions, in particular during the first step of pathogen recognition. In this study we present the chemical structure of OPS of the first Pectobacterium aquaticum strain (IFB5637) isolated from water in Poland. The OPS consists of two common hexoses, such as mannose and glucose, as well as an abequose (3,6-dideoxy-d-xylo-hexose), the first 3,6-dideoxyhexose identified among the Pectobacteriaceae family: According to our best knowledge this is the first determined structure of the OPS of P. aquaticum.

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.

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.

Microbe Profile: Pectobacterium atrosepticum: an enemy at the door.

Pectobacterium atrosepticum is part of a larger family of soft rot bacteria (Pectobacteriaceae) that cause disease on a wide range of crops worldwide. They are closely related to members of the Enterobacteriaceae and, as the plant pathogens and plant associated members of the group, form part of a continuum towards opportunistic and more devastating animal and human pathogens. Many of the horizontally acquired islands present in the genome of P. atrosepticum are directly responsible for life on plants. These include genes for a plethora of plant cell wall degrading enzymes, plant toxins, siderophores etc., which are exported by multiple secretion systems under a highly coordinated regulation system.

Insights into complex infection by two Pectobacterium species causing potato blackleg and soft rot.

Pectobacterium spp. are causative agents of blackleg and soft rot of potato. However, little is known about the relationship between the pathogenicity of mixed infections of different Pectobacterium spp. at different temperatures. In this study, two pectinolytic strains of Pectobacterium spp. were isolated from the same potato plant with typical symptoms of blackleg and identified as P. brasiliense and P. carotovorum by multilocus sequence analysis (MLSA), whole-genome phylogenetic tree construction, average nucleotide identity (ANI) analysis and digital DNA-DNA hybridization (dDDH). Plant cell wall degrading enzyme, including pectinases, cellulases and proteases, as the most important virulence factors, as well as pathogenicity toward potato tuber, were compared between the strains P. brasiliense BL-2 and P. carotovorum BL-4 at 28 â„ƒ. The results showed that P. carotovorum had higher cell wall-degrading enzyme activities and brought more severe disease symptoms to potato tubers than P. brasiliense. Moreover, the pathogenicity of P. carotovorum and P. brasiliense increased with increasing temperature (20, 25, 28, 32 â„ƒ). The pathogenicity was more severe when P. carotovorum strain BL-4 was co-inoculated with P. brasiliense strain BL-2, especially when the former exhibited an advantage in bacterial number at the initial time. The results of this study provide new insight for understanding the pathogenicity caused by mixed infections with different species of Pectobacterium spp., and they may provide some guidance for controlling potato blackleg and soft rot.

Whole-cell bacterial biosensor for volatile detection from Pectobacterium-infected potatoes enables early identification of potato tuber soft rot disease.

Half of the harvested food is lost due to rots caused by microorganisms. Plants emit various volatile organic compounds (VOCs) into their surrounding environment, and the VOC profiles of healthy crops are altered upon infection. In this study, a whole-cell bacterial biosensor was used for the early identification of potato tuber soft rot disease caused by the pectinolytic bacteria Pectobacterium in potato tubers. The detection is based on monitoring the luminescent responses of the bacteria panel to changes in the VOC profile following inoculation. First, gas chromatography-mass spectrometry (GC-MS) was used to specify the differences between the VOC patterns of the inoculated and non-inoculated potato tubers during early infection. Five VOCs were identified, 1-octanol, phenylethyl alcohol, 2-ethyl hexanol, nonanal, and 1-octen-3-ol. Then, the infection was detected by the bioreporter bacterial panel, firstly measured in a 96-well plate in solution, and then also tested in potato plugs and validated in whole tubers. Examination of the bacterial panel responses showed an extensive cytotoxic effect over the testing period, as seen by the elevated induction factor (IF) values in the bacterial strain TV1061 after exposure to both potato plugs and whole tubers. Moreover, quorum sensing influences were also observed by the elevated IF values in the bacterial strain K802NR. The developed whole-cell biosensor system based on bacterial detection will allow more efficient crop management during postharvest, storage, and transport of crops, to reduce food losses.

Host range and virulence diversity of Pectobacterium carotovorum subsp. brasiliense strain RDKLR infecting radish in India, and development of a LAMP-based diagnostics.

AIM: This work aimed at determining the pathogenicity, molecular characterization, host range and rapid detection of Pectobacterium carotovorum subsp. brasiliense (Pcb) causing soft rot disease in radish. METHODS AND RESULTS: The four isolated isolates were inoculated to radish, typical soft rot symptoms were observed and Koch's postulates were proved. The most virulent strain RDKLR was morphologically and biochemically distinct. Pcb showed a positive potato soft rot test and elicited hypersensitivity response on Nicotiana tobaccum. The genes Pel2 and pmrA were used for subspecies characterization of Pcb. It has a wide host range and infection was observed on slices of carrot, tomato, radish, potato, cauliflower, cabbage, chilli, knol-khol, bell pepper and cucumber. Infectivity was also seen in seedlings under glasshouse conditions. Pcb produced cell wall degrading enzymes in semi-quantification assay and is a strong biofilm producer. The LAMP technique was standardized to help rapid detection and take prophylactic measures to manage the disease. CONCLUSION: This work reports Pcb as a new soft rot causing organism of radish in India. Pcb is highly virulent with a broad host range. The LAMP technique helps in rapid detection. SIGNIFICANCE AND IMPACT OF THE STUDY: Pcb-induced soft rot causes significant yield loss, decreased market value, damage in transit, storage and the market. Disease characterization and early identification aid in disease management and prevention in the field.

Development of a multiplex TaqMan qPCR targeting unique genomic regions for the specific and sensitive detection of Pectobacterium species and P. parmentieri.

AIM: The newly defined species Pectobacterium parmentieri has emerged as an aggressive pathogen that causes soft rot and blackleg diseases on potato and has been widely disseminated across the globe, jeopardizing the productivity and potato food safety. The implementation of a fast and accurate detection tool is imperative to control, monitor and prevent further spread of these pathogens. The objective of this work was to develop a specific and sensitive multiplex TaqMan qPCR to detect P. parmentieri and distinguish it from all known Pectobacterium species. A universal internal control was included to enhance the reliability of the assay. METHODS AND RESULTS: A comparative genomics approach was used to identify O-acetyltransferase and the XRE family transcriptional regulator as specific targets for primers/probe design for the detection of the Pectobacterium genus and P. parmentieri, respectively. Specificity was assessed with 35 and 25 strains included in the inclusivity and exclusivity panels, respectively, isolated from different geographical locations and sources. The assay specifically detected all 35 strains of Pectobacterium sp. and all 15 P. parmentieri strains. No cross-reactivity was detected during assay validation. Our assay detected up to 10 fg genomic DNA and 1 CFU ml-1 bacterial culture. No change in the detection threshold (1 CFU ml-1 ) was observed in spiked assays after adding host tissue to the reactions. The assay was validated with naturally and artificially infected host tissues and soil rhizosphere samples. All infected plant samples containing the target pathogens were accurately amplified. CONCLUSION: The presented multiplex TaqMan qPCR diagnostic assay is highly specific, sensitive, reliable for the detection of Pectobacterium species and P. parmentieri with no false positives or false negatives. SIGNIFICANCE AND IMPACT OF THE STUDY: The developed assay can be adopted for multiple purposes such as seed certification programmes, surveillance, biosecurity, microbial forensics, quarantine, border protection, inspections and epidemiology.

Phage Therapy for Crops: Concepts, Experimental and Bioinformatics Approaches to Direct Its Application.

Phage therapy consists of applying bacteriophages, whose natural function is to kill specific bacteria. Bacteriophages are safe, evolve together with their host, and are environmentally friendly. At present, the indiscriminate use of antibiotics and salt minerals (Zn2+ or Cu2+) has caused the emergence of resistant strains that infect crops, causing difficulties and loss of food production. Phage therapy is an alternative that has shown positive results and can improve the treatments available for agriculture. However, the success of phage therapy depends on finding effective bacteriophages. This review focused on describing the potential, up to now, of applying phage therapy as an alternative treatment against bacterial diseases, with sustainable improvement in food production. We described the current isolation techniques, characterization, detection, and selection of lytic phages, highlighting the importance of complementary studies using genome analysis of the phage and its host. Finally, among these studies, we concentrated on the most relevant bacteriophages used for biocontrol of Pseudomonas spp., Xanthomonas spp., Pectobacterium spp., Ralstonia spp., Burkholderia spp., Dickeya spp., Clavibacter michiganensis, and Agrobacterium tumefaciens as agents that cause damage to crops, and affect food production around the world.