1.3.34
DEVELOPMENT OF A RAPID ASSAY TO DETERMINE VIABILITY OF BOTRYTIS CINEREA PROTOPLASTS

NJ SPICE1, WR CARLILE1 and AH COBB2

1Department of Life Sciences, Nottingham Trent University, Clifton Lane, Nottingham NG11 8NS, UK; 2Crop and Environmental Research Centre, Harper Adams Agricultural College, Newport, Shropshire TFIO 8NB, UK

Background and objectives
Fluorochromes are dyes that fluoresce when excited by ultraviolet (UV) or visible light. The determination of cell viability, which allows live cells to be distinguished from dead cells within a population, can be achieved using fluorescent dyes either alone or in combination. Cell permeant fluorogenic esterase substrates, such as fluorescein diacetate (FDA), act as viability probes and are capable of determining both enzyme activity and membrane integrity of living cells [1]. FDA has previously been used to determine the viability of plant, fungal and red algal protoplasts and is a useful indicator of percentage viability. In vitro assays allowing the quantification of fluorescent intensity have been successfully used in mammalian and plant cells, plant protoplasts and viral agents. The development of an assay allowing quantification of the fluorescence from FDA-treated fungal protoplasts using a fluorescence spectrophotometer may provide a means of rapidly screening protoplasts treated with potential toxophores for viability, as well as a means of evaluating protoplast sensitivity to other compounds including organic solvents and adjuvants.

Materials and methods
Protoplasts were isolated from B. cinerea as described previously [2] and incubated with FDA prior to measurement of fluorescence. A number of experimental parameters, including FDA concentration, incubation period prior to detection of fluorescence, protoplast concentration and background fluorescence were investigated. The sensitivity of the assay was then investigated by challenging the protoplasts with treatments expected to significantly alter their viability prior to addition of FDA. The tolerance of protoplasts to varying concentrations of a range of organic solvents was also determined.

Results and conclusions
Optimal assay conditions were determined. It was found that incubation of protoplasts with 0.1 g or 1 PM FDA produced minimal fluorescence, whilst incubation with 10 g or IOO PM FDA resulted in an increased fluorescence; 10 MM FDA was optimal. The extent of fluorescence detected increased over the time period investigated, as the protoplasts cleaved the FDA and the fluorescein accumulated within the viable intact cells. A 10-min incubation period was optimal. A significant (P<0.01) positive correlation between protoplast concentration and fluorescence intensity was observed. Protoplast concentrations between 5x105 and 1x106 per ml were optimal. Low background fluorescence was detected from the protoplasts, buffer and FDA stain. Protoplasts treated with 0.1 N HCl, 0.01 M C8 surfactant or heat did not fluoresce, probably due to both a lack of esterase activity and poor membrane integrity. The tolerance of protoplasts to methanol, ethanol, acetone and dimethylsulfoxide was investigated. Protoplasts were able to withstand incubation in concentrations of 1% (v/v) solvent over the time period investigated. Viability as determined by the assay was significantly reduced by incubation in 10% (v/v) solvent. The potential uses of the assay are discussed.

References
1. Haughland RP, 1996. Handbook of Fluorescent Probes and Research Chemicals, 6th Edn. Molecular Probes Inc., Eugene, Oregon, USA.
2. Spice NJ, Carlile WR, Cobb AH, 1996. Proceedings Brighton Crop Protection Conference: Pests and Diseases, pp. 433-434.