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Development of antibody to virulence factor flagellin and its evaluation in screening Ralstonia pseudosolanacearum

  • Soil and Agricultural Microbiology - Research Paper
  • Published:
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Abstract

The bacterial wilt disease caused by Ralstonia pseudosolanacearum presents a notable economic risk to a variety of crucial crops worldwide. During preliminary isolation of this phytopathogen, several colonies of other saprophytic bacteria may be mistaken with it. So, the present study aims to address this issue by proposing the application of immunogenic proteins, particularly flagellin (FliC), to enable a rapid and early identification of bacterial wilt. In this study, a novel approach is unveiled for the early detection of R. pseudosolanacearum. The study exploits the immunogenic attributes of flagellin (FliC), by generating polyclonal antibodies against recombinant FliC within model organisms—rabbits and mice. The efficacy of these antibodies is meticulously assessed through discerning techniques, including DAS-ELISA and Western blot analyses, which elucidate their remarkable specificity in identifying various R. pseudosolanacearum strains. Furthermore, the introduction of antibody-coated latex agglutinating reagents offers an additional layer of confirmation, substantiating the feasibility of establishing a laboratory-based toolkit for swift screening and unambiguous identification of the bacterial wilt pathogen. This study presents a significant stride toward enhancing early diagnostic capabilities, potentially revolutionizing agricultural practices by safeguarding crop yield and quality through proactive pathogen detection and mitigation strategies.

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Abbreviations

BSA:

Bovine serum albumin

CFU:

Colony-forming unit

CPG:

Casamino acid-peptone-glucose

DAB:

3,3′-Diaminobenzidine

DAS-ELISA:

Double-antibody sandwich enzyme-linked immunosorbent assay

DVC:

Direct viable count

EDAC:

1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide

ELISA:

Enzyme-linked immunosorbent assay

FliC:

Flagellin

FP:

Forward primer

His:

Histidine

HRP:

Horseradish peroxide

H202:

Hydrogen peroxide

IgG:

Immunoglobulin G

IPTG:

Isopropyl β-d-1-thiogalactopyranoside

LAT:

Latex agglutination test

MES:

2-morpholino ethane sulfonic acid

mSMSA:

Modified semi-selective medium South Africa

Ni-NTA:

Nickel-nitrilotriacetic acid

PBS:

Phosphate-buffered saline

PCR:

Polymerase chain reaction

PVDF:

Ppolyvinylidene difluoride

RP:

Reverse primer

RPS:

Ralstonia pseudosolanacearum

RS:

Ralstonia solanacearum

RSSC:

Ralstonia solanacearum species complex

RPM:

Revolutions per minute

SDS-PAGE:

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis

WC:

Whole cell

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Acknowledgements

The authors are very grateful to the DIBER-DRDO, Haldwani, Uttarakhand, India, and PP Savani University, Surat, Gujarat, India, for providing all the necessary assistance during the study and compilation of the work. The authors are also thankful to Dr. Rakshit Pathak and Mr. Mayank Punetha for the graphics and technical support.

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Authors and Affiliations

  1. P P Savani University, Surat, Gujarat, 394125, India

    Shalini Bhatt

  2. Defence Institute of Bio-Energy Research (DIBER), DRDO, Haldwani, Nainital, Uttarakhand, 263139, India

    Shalini Bhatt, S. Merwyn P. Raj, Neha Faridi, Dinesh Pathak, Ankur Agarwal & Shraddha P. Mishra

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  1. Shalini Bhatt
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  2. S. Merwyn P. Raj
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Contributions

Study conceptualization: Shalini Bhatt, S. Merwyn P. Raj; methodology: Shalini Bhatt, S. Merwyn P. Raj, Neha Faridi; formal analysis and investigation: Shalini Bhatt, S. Merwyn P. Raj, Shraddha Mishra; writing—original draft preparation: Shalini Bhatt; writing—review and editing: Shalini Bhatt; supervision: Ankur Agarwal, S. Merwyn.

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Correspondence to Shalini Bhatt.

Ethics declarations

Polyclonal antibodies used in the study were outsourced by Abgenex Private Limited, Bhubaneswar, India.

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The authors declare no competing interests.

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Bhatt, S., Raj, S.M.P., Faridi, N. et al. Development of antibody to virulence factor flagellin and its evaluation in screening Ralstonia pseudosolanacearum. Braz J Microbiol 55, 809–821 (2024). https://doi.org/10.1007/s42770-023-01235-4

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