BY

EUGENE MADZOKERE MOLECULAR BIOLOGY SERVICES CHINHOYI UNIVERSITY OF TECHNOLOGY

Control of Rhizoctonia solani species and root-knot nematodes using chitinolytic and antibiotic producing Pseudomonas species

Structure of Presentation

Introduction of the problem

Methods

Application

Benefits of Proposed Controls

Conclusion & Recommendations

Acknowledgements

Introduction

R. solani disease symptoms oLeaf Spot oStem & root rot oDamping-off oSore shin

Nematode disease symptoms oRoot galls oStunted growth oChlorosis

Identification of specific Pseudomonas species

I. Suppressive soils e.g. P. fluorescens, P. chlororaphis, & P. aeruginosa

II. Morphological: Colloidal Chitin test

III. Spectral: fluorescence at 365nm

IV. Molecular:

a. PCR with pseudomonas specific primers :

i. PsF/PsR

b. PCR with antibiotic specific primers :

i. PRN - pyrrolnitrin

ii. Phl2a/Phl2b - 2, 4-diacetylphloroglucinol &

iii. PCA - phenazine-1-carboxylic acid

Polyhedrin promoter

Plasmid transfer vector e.g. PVL 1393/ PAcAB3

Co-transfection into expressSF+ cells

Transfer expressSF+ cells for fermentation in Serum-free medium

Scale-up of fermentation for harvesting high antibiotic yield

Filtration & harvesting of cell-free extract

Ethyl acetate extraction of Antibiotics

Detection & Purification of Antibiotics by Chromatography

e

Production of Antibiotics Using the ProEasyTM BEVS Technology

Antibiotic encoding gene

Linearized ProEasy Tm

Baculovirus DNA

Production of Inoculum

Batch culture within Kings-B Broth

24-36 hours to harvest

Cell densities high hence high inoculum yield

Application of Controls

Bacterial suspension & antibiotic solutions as root dip & drenches

Soil inoculants at transplanting & after

Low cost carriers e.g. peat, calcined clay, powdered corn cobs, alginate microbeads

50-94% pathogen inhibition efficacy reported against AG 2-2, AG-3, AG-4, AG-5 R. solani species and Meloidogyne incognita species

Cameroon, USA, Netherlands, India, Australia, South Korea, China, Canada successfully controlled pathogenic species

Broad spectral action of controls

Broad spectral action of controls continued…

Benefits of Technology

Promotes healthy plant growth via nutrient uptake, root growth & induced systemic resistance (ISR)

High quality antibiotic yield

Production processes highly scalable

High recombinant viral titer & cell densities

Control is non-phytopathogenic, environmentally safe & cost efficient.

Applicable with tobacco, rice, wheat, bean, potato, & citrus fruits

Conclusion

Efficient control of Rhizoctonia solani and root-knot nematode species demands an integrated management approach.

Making use of

i. High efficacy fungicides, nematicides, biocontrols & prudent agric-practices

ii. Environmentally safe agrochemical controls

Recommendations

I. Consider application of Pseudomonas species inoculants & antibiotic products as broad spectrum controls

II. Adopt alternative screening techniques for Chitinolytic activity within antagonistic soil-borne microflora

III. Produce and commercialize the 3 antibiotics & Pseudomonas Inoculants

Acknowledgements

Dr Garwe

Mr. Muzhinji

Plant Health Clinic (PHC) Personnel

MBS, PB & PHC Students

THANK YOU