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
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