Antinemic activity of Plant Growth Promoting Rhizobacteria, Pseudomonas fluorescens against root knot nematode, Meloidogyne incognita infesting tomato, Lycopersicon esculentum Mill.

Table of Contents

Random survey for the isolation of PGPR in Tamil Nadu

Plant growth promotion

i. Roll towel studies

ii. Pot culture method

Biochemical characterization of Pseudomonas strains

(i). Gram staining

(ii). Potassium hydroxide (KOH) test

(iii). Catalase test

(iv). Starch hydrolysis

(iv). Gelatin hydrolysis

(v). Methyl red test

(vi). Hydrogen cyanide (HCN) production

(vii). Siderophore production

Molecular characterization of the Pseudomonas strains

Detection of Pseudomonas species specific loci in the rhizobacterial strains

Detection of antibiotics produced by Pseudomonas fluorescens in Thin Layer Chromatography (TLC)

(i). Detection of 2, 4-DAPG

(ii). Detection of pyoluteorin

(iii). Detection of Phenazine (Phenazine 1-carboxylic acid)

(iv). Detection of Pyocyanine

Detection of antibiotics produced by P. fluorescens by Polymerase Chain Reaction

PCR based detection of 2,4-diacetylphloroglucinol (DAPG) and phenazine carboxylic acid (PCA) genes in fluorescent pseudomonads

Identification of root knot nematode species associated with tomato

Extraction of crude antibiotics from P. fluorescens strains

In vitro efficacy of crude antibiotics of P. fluorescens against M. incognita

Hatching and Mortality studies

Developing liquid formulation of P. fluorescens strains

Developing liquid formulation of P. fluorescens isolates

Efficacy of P. fluorescens strains against nematodes infesting tomato under pot culture condition

Split root bioassay

Efficacy of P. fluorescens strains against nematodes infesting tomato under field condition

Evaluating the liquid based formulation of P. fluorescens isolates in tomato against M. incognita under field conditions

Induction of defense related enzymes in Pseudomonas treated plants by native - PAGE analysis

Assessment of proteins associated with defense activities due to application of Pseudomonas isolates by Sodium Dodecyl Sulphate Polyacrylamide gel electrophoresis (SDS-PAGE)

Statistical analysis

Research Objectives and Focus Areas

The primary research objective is to isolate and evaluate native strains of Pseudomonas fluorescens for their biological control efficacy against the root-knot nematode Meloidogyne incognita in tomato, specifically focusing on the development of stable liquid formulations.

• Isolation and biochemical/molecular characterization of Pseudomonas fluorescens strains.
• Evaluation of antibiotic production (2,4-DAPG, pyoluteorin, etc.) via TLC and PCR.
• In vitro assessment of crude antibiotic extracts on nematode hatching and juvenile mortality.
• Pot culture and field trial evaluations of liquid formulations of P. fluorescens against M. incognita.
• Analysis of plant defense-related enzymes (PO, PPO) and protein induction in treated tomato plants.

Excerpt from the Book

Summary of Chapters

Random survey for the isolation of PGPR in Tamil Nadu: Describes the collection of 35 PGPR strains from major tomato-growing districts in Tamil Nadu during 2007-2008.

Plant growth promotion: Details the methodologies for assessing plant growth promotion using roll towel and pot culture techniques.

Biochemical characterization of Pseudomonas strains: Outlines the laboratory procedures for confirming the bacterial genus through various biochemical tests like Gram staining, catalase, and hydrolysis assays.

Molecular characterization of the Pseudomonas strains: Explains the PCR-based confirmation of Pseudomonas species using specific 16S-23S rRNA primers.

Detection of antibiotics produced by Pseudomonas fluorescens in Thin Layer Chromatography (TLC): Discusses the chemical extraction and verification of secondary metabolites such as 2,4-DAPG, pyoluteorin, and phenazine.

Detection of antibiotics produced by P. fluorescens by Polymerase Chain Reaction: Details the detection of specific biosynthetic loci for antibiotic production using specialized DNA primers.

Identification of root knot nematode species associated with tomato: Explains the morphological identification of Meloidogyne incognita based on perenial area cuticular markings.

Extraction of crude antibiotics from P. fluorescens strains: Describes the technical process of acid precipitation and solvent extraction to obtain concentrated extracellular compounds.

In vitro efficacy of crude antibiotics of P. fluorescens against M. incognita: Reports on the impact of crude antibiotic extracts on egg mass hatching and juvenile mortality rates.

Developing liquid formulation of P. fluorescens strains: Covers the creation of liquid formulations using King's B broth and glycerol to facilitate agricultural application.

Efficacy of P. fluorescens strains against nematodes infesting tomato under field condition: Presents the results of field trials conducted in Coimbatore to evaluate the liquid formulation's performance in real-world settings.

Induction of defense related enzymes in Pseudomonas treated plants by native - PAGE analysis: Investigates the systemic biochemical changes, specifically Peroxidase (PO) and Polyphenoloxidase (PPO) activity, in treated plants.

Assessment of proteins associated with defense activities due to application of Pseudomonas isolates by Sodium Dodecyl Sulphate Polyacrylamide gel electrophoresis (SDS-PAGE): Analyzes the induction of specific defense-related proteins in tomato roots following bacterial application.

Keywords

Pseudomonas fluorescens, Meloidogyne incognita, Tomato, Root-knot nematode, Liquid formulation, Biological control, Plant growth promoting rhizobacteria, Antibiotics, Induced systemic resistance, 2,4-DAPG, PCR, Peroxidase, Polyphenoloxidase, Siderophore, Biocontrol

Frequently Asked Questions

What is the core focus of this research?

The research focuses on utilizing Pseudomonas fluorescens as a biocontrol agent against Meloidogyne incognita in tomato plants, specifically through the development and testing of effective liquid-based formulations.

What are the primary thematic areas covered?

The work covers bacterial isolation, biochemical and molecular characterization, antibiotic synthesis assessment, in vitro and in vivo efficacy trials against nematodes, and the induction of plant defense mechanisms.

What is the primary objective or research question?

The main goal is to determine if liquid formulations of native P. fluorescens strains can effectively reduce nematode infestations and improve growth and yield in tomato crops compared to traditional control methods.

Which scientific methods are employed?

The study employs a multi-disciplinary approach, including microbial isolation, PCR and TLC analysis, split-root bioassays, field trials, and protein electrophoresis (PAGE/SDS-PAGE) to measure biochemical resistance.

What is addressed in the main part of the work?

The main sections document the entire pipeline from isolating beneficial bacteria in Tamil Nadu, verifying their biocontrol properties, confirming their genomic potential, and proving their efficacy in pot culture and field conditions.

Which keywords characterize this work?

Keywords include Pseudomonas fluorescens, Meloidogyne incognita, biocontrol, tomato, liquid formulation, induced systemic resistance, and secondary metabolites.

Which specific P. fluorescens strains were identified as most effective?

Strain Pf 128 consistently demonstrated the highest efficacy in most trials, including superior root colonization, highest reduction in nematode populations, and the highest fruit yield in field evaluations.

How does the liquid formulation contribute to nematode suppression?

The formulation helps by establishing the bacteria in the rhizosphere, which then produces secondary metabolites (antibiotics) and triggers the plant's own systemic resistance mechanisms (ISR), resulting in reduced gall formation and nematode reproduction.

What role do giant cells play in the host-parasite relationship mentioned?

Giant cells are specialized feeding sites developed by the host plant at the site of nematode infection; they act as transfer cells to provide necessary nutrients to the parasitic nematode.

Why is the split-root bioassay significant in this study?

The split-root bioassay allows researchers to test treatments on one half of the root system while keeping the other half as a control, effectively minimizing environmental variations and proving the systemic nature of the induced plant resistance.