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Title: Evaluating the use of fungal protoplasts to investigate fungicide action
Author: Spice, Nicola Jane
ISNI:       0000 0001 3475 1927
Awarding Body: Nottingham Trent University
Current Institution: Nottingham Trent University
Date of Award: 2000
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Protoplasts have been successfully isolated from 3 economically important plant pathogenic fungi, Botrytis cinerea, Phytophthora infestans and Stagonospora nodorum, and characterised with respect to their metabolic capabilities using fluorescent microscopy and polarography techniques. The effects of a series of linear alcohol ethoxylate surfactants upon respiration in protoplasts of B. cinerea have been examined. The length of the carbon chain within the molecules, rather than the ability of the surfactants to form micelles in solution was shown to be the predominant driving force leading to membrane disruption. The smaller molecules (C8 & C12) were able to solubilize the protoplast membrane, whilst the larger molecule (C16) did not cause protoplasts to rupture at the concentrations tested. The preferential carbon chain length for protoplast mteraction was found to be C8, which caused membrane solubilization at 4mM, but not at 2mM. The fluorescent dye fluorescein diacetate was successfully used spectrophotometerically to determine the viability of B. cinerea protoplasts. Concentrations of between 5x10⁵ and 1x10⁶ protoplasts ml⁻¹ incubated with 10μM FDA over a 10min time period were optimal. Low levels of background fluorescence could be accounted for using simple mathematical formulae. Protoplasts subjected to treatments expected to significantly reduce their viability showed a corresponding reduction in FDA-derived fluorescence. The assay was used to determine protoplast tolerance of different organic solvents. Protoplasts were found to tolerate acetone, ethanol, methanol and DMSO at concentrations up to and including 1% (v/v aq.). DMSO and methanol did not significantly reduce protoplast viability, even at 10%. The effects of 2 commercially available fungicides on protoplast fluorescence were investigated. Incubation with azoxystrobin was found to significantly reduce FDA derived fluorescence at 10 and 100μM (at 30 and 60min) and at 1μM (60min only). Incubation with pyrimethanil did not cause a significant reduction in protoplast viability, which could he linked to the mode of action of this compound. Radiolabelled amino acids were incorporated into proteins within viable protoplasts, but not non-viable protoplasts of B. cinerea. A suitable assay was developed for the detection of this incorporation. A preferential growth medium which stimulated protoplast regeneration and hence protein synthesis was determined. Protoplast association with the radiolabelled experimental fungicide "AG1" was found to he higher in non-viahle protoplasts. A high percentage of the compound was found to he associated with the protoplast samples, probably linked to the lipophilicity of the compound. An assay was developed and optimised to investigate the association of a range of radiolabelled compounds with B. cinerea protoplasts. Compounds were chosen with reference to log P values. A relationship between compound log P value and association with a protoplast population was discovered; association of the compound with the protoplasts increased with increasing log P values. A "standard curve" was generated by the assay, which could potentially provide a useful marker to study structure optimisation and delivery of radiolabelled experimental compounds. The effectiveness of known and experimental fungicides against B. cinerea was investigated using a variety of assays, in order to establish the usefulness of protoplasts in this context. Large discrepancies were discovered between compound effectiveness in planta, where no compound offered good protection against B. cinerea infection, and in vitro where most compounds inhibited the pathogen to various extents depending on the assays employed (measurement of growth on agar, oxygen uptake by germlings and protoplasts, and cytochrome c reduction in the electron transport chain of isolated mitochondria). Potentially useful applications of protoplast technology within the agrochemical industry have been discussed.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: Agricultural chemistry & fertilizers