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Detection of the Plant Pathogen Pseudomonas Syringae pv. Lachrymans on Antibody-Modified Gold Electrodes by Electrochemical Impedance Spectroscopy.

ABSTRACT: The present work describes an impedimetric immunosensor for Pseudomonas syringae pv. lachrymans (Psl) detection. This pathogen infects many crop species causing considerable yield losses, thus fast and cheap detection method is in high demand. In the assay, the gold disc electrode was modified with 4-aminothiophenol (4-ATP), glutaraldehyde (GA), and anti-Psl antibodies, and free-sites were blocked with bovine serum albumin (BSA). Sensor development was characterized by cyclic voltammetry (CV) and antigen detection by electrochemical impedance spectroscopy (EIS) measurements. Seven analyzed strains of Psl were verified as positive by the reference method (PCR) and this immunoassay, proving sensor specificity. Label-free electrochemical detection was in the linear range 1 × 103-1.2 × 105 CFU/mL (colony-forming unit) with an R2 coefficient of 0.992 and a detection limit (LOD) of 337 CFU/mL. The sensor did not interfere with negative probes like buffers and other bacteria. The assay was proven to be fast (10 min detection) and easy in preparation. The advantage was the simplicity and availability of the verified analyte (whole bacteria) as the method does not require sample pretreatment (e.g., DNA isolation). EIS biosensing technique was chosen as one of the simplest and most sensitive with the least destructive influence on the probes compared to other electrochemical methods.

SUBMITTER: Cebula Z

PROVIDER: S-EPMC6960962 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:The Pseudomonas syringae complex comprises different genetic groups that include strains from both agricultural and environmental habitats. This complex group has been used for decades as a "hodgepodge," including many taxonomically related species. More than 60 pathovars of P. syringae have been described based on distinct host ranges and disease symptoms they cause. These pathovars cause disease relying on an array of virulence mechanisms. However, P. syringae pv. syringae (Pss) is the most polyphagous bacterium in the P. syringae complex, based on its wide host range, that primarily affects woody and herbaceous host plants. In early 1990s, bacterial apical necrosis (BAN) of mango trees, a critical disease elicited by Pss in Southern Spain was described for the first time. Pss exhibits important epiphytic traits and virulence factors, which may promote its survival and pathogenicity in mango trees and in other plant hosts. Over more than two decades, Pss strains isolated from mango trees have been comprehensively investigated to elucidate the mechanisms that governs their epiphytic and pathogenic lifestyles. In particular, the vast majority of Pss strains isolated from mango trees produce an antimetabolite toxin, called mangotoxin, whose leading role in virulence has been clearly demonstrated. Moreover, phenotypic, genetic and phylogenetic approaches support that Pss strains producers of BAN symptoms on mango trees all belong to a single phylotype within phylogroup 2, are adapted to the mango host, and produce mangotoxin. Remarkably, a genome sequencing project of the Pss model strain UMAF0158 revealed the presence of other factors that may play major roles in its different lifestyles, such as the presence of two different type III secretion systems, two type VI secretion systems and an operon for cellulose biosynthesis. The role of cellulose in increasing mango leaf colonization and biofilm formation, and impairing virulence of Pss, suggests that cellulose may play a pivotal role with regards to the balance of its different lifestyles. In addition, 62-kb plasmids belonging to the pPT23A-family of plasmids (PFPs) have been strongly associated with Pss strains that inhabit mango trees. Further, complete sequence and comparative genomic analyses revealed major roles of PFPs in detoxification of copper compounds and ultraviolet radiation resistance, both improving the epiphytic lifestyle of Pss on mango surfaces. Hence, in this review we summarize the research that has been conducted on Pss by our research group to elucidate the molecular mechanisms that underpin the epiphytic and pathogenic lifestyle on mango trees. Finally, future directions in this particular plant-pathogen story are discussed.

Project description:Mobile genetic elements are widespread in Pseudomonas syringae, and often associate with virulence genes. Genome reannotation of the model bean pathogen P. syringae pv. phaseolicola 1448A identified seventeen types of insertion sequences and two miniature inverted-repeat transposable elements (MITEs) with a biased distribution, representing 2.8% of the chromosome, 25.8% of the 132-kb virulence plasmid and 2.7% of the 52-kb plasmid. Employing an entrapment vector containing sacB, we estimated that transposition frequency oscillated between 2.6×10(-5) and 1.1×10(-6), depending on the clone, although it was stable for each clone after consecutive transfers in culture media. Transposition frequency was similar for bacteria grown in rich or minimal media, and from cells recovered from compatible and incompatible plant hosts, indicating that growth conditions do not influence transposition in strain 1448A. Most of the entrapped insertions contained a full-length IS801 element, with the remaining insertions corresponding to sequences smaller than any transposable element identified in strain 1448A, and collectively identified as miniature sequences. From these, fragments of 229, 360 and 679-nt of the right end of IS801 ended in a consensus tetranucleotide and likely resulted from one-ended transposition of IS801. An average 0.7% of the insertions analyzed consisted of IS801 carrying a fragment of variable size from gene PSPPH_0008/PSPPH_0017, showing that IS801 can mobilize DNA in vivo. Retrospective analysis of complete plasmids and genomes of P. syringae suggests, however, that most fragments of IS801 are likely the result of reorganizations rather than one-ended transpositions, and that this element might preferentially contribute to genome flexibility by generating homologous regions of recombination. A further miniature sequence previously found to affect host range specificity and virulence, designated MITEPsy1 (100-nt), represented an average 2.4% of the total number of insertions entrapped in sacB, demonstrating for the first time the mobilization of a MITE in bacteria.

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Detection of the Plant Pathogen Pseudomonas Syringae pv. Lachrymans on Antibody-Modified Gold Electrodes by Electrochemical Impedance Spectroscopy.

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