Detection of Viable Ascospores of Neosartorya

Abstract

MATERIALS AND METHODS Studies on 29 Neosartorya cultures showed that many as-cospores, as enumerated in a haemocytometer, failed to produce colonies when cultured on agar media. Although the percent recoveries differed greatly between spore crops, germination and out-growth of most were stimulated by heat activation in grape juice for 30-60 min at 70°C. Most probable number and CFU counts were usually similar which indicated that low recoveries were not due to an inadequate incubation period or to the production of self-inhibitory compounds. Low percent recoveries did not affect the D values obtained in heat-resistance trials. The ascospores of Neosartorya are sufficiently heat resistant to survive the commercial thermal processes that are given to juices and other fruit products (2,3,9). Increasing the severity of the process often is not a feasible solution because of carmelization and other adverse effects on food quality. Many processors minimize the incidence of spoilage by monitoring fruit and various ingredients for heat-resistant spores. To do this effectively, sensitive methods for the detection of spores, many which are in a dormant state, must be used. Heat shock often is required before ascospores will germinate and produce vegetative growth. Strains appear to differ in the amount of heat required for activation: the optimal time and temperature for Neosartorya fischeri FRR-1265 was 45 min at 70°C, while strain FRR-2334 required 15 min at 85°C (7). The heating medium also influences activation. Thus, Aspergillus WR-1 (Neosartorya) yielded significantly higher recoveries when heated in grape and other fruit juices than when heated in water (77). High concentrations of spores sometimes germinate less well than low concentrations (4,72). This can be due to self-inhibitors of spore germination which are produced by some fungi. These inhibitors include both volatile and nonvolatile compounds (7). Acetaldehyde, n-butyraldehyde, n-propanol, and ethyl acetate are examples of compounds that influence the recovery of spores (6). In this investigation, 29 strains and species of Neosartorya were studied to determine some of the factors that affect the detection of viable spores. The effect of different variables on germination and outgrowth was assessed by comparing microscopic ascospore counts with colony counts on agar media. Test organisms The cultures were isolated from a number of sources with the majority originating from temperate and tropical soils. Six of the isolates had been responsible for spoilage outbreaks involving thermally processed fruit products. The taxonomic position of the two isolates designated N. sp. nov. 1 and N. sp. nov. 2 could not be determined. This work is still in progress. Three ascospore crops were studied. Crop 1 was harvested from cultures grown 3-4 weeks at 30°C on oat meal agar (8). Aqueous suspensions were prepared by scraping the plates with sterile glass rods (5). The suspensions were treated in 2-min increments in an ice-cooled sonicator water bath to break up clumps and asci. The ascospores were then stored in water at 5°C. Crop 2 ascospores were grown on a medium containing Bacto malt extract, 30 g; agar, 15 g; distilled water, 1 L; pH 5.5. The spores were harvested by scraping the plates after an incubation of 30 d at 30°C The aqueous suspensions were treated 20 min in a Bransonic water bath sonicator (SmithKline, Philadelphia) to break up clumps and asci. The ascospores were stored at-15°C. Crop 3 spores were treated in a manner identical to the Crop 2 ascospores except that the cultures were grown on oat meal-wheat germ agar (7). Heat activation In a typical trial, 0.1 ml of stock ascospore suspension was pipetted into a 1.5-ml microcentrifuge tube. After the mixing in 0.9 ml of 95% ethanol (to destroy conidia and hyphae), the suspension was centrifuged 5 min at 14,000 x g. The liquid was then decanted, and the ascospores were resuspended in 1.0 ml of activation media. The centrifuge tubes containing the suspensions were stored in shaved ice until they could be transferred to a stirred water bath that had been equilibrated to the desired temperature. Following activation , appropriate decimal dilutions were made in sterile water. Ascospore enumeration Direct microscopic counts were determined with a haemocytometer. Ascospores were readily recognizable at 100-fold magnification due to their characteristic equatorial crests. Bacto potato dextrose agar, pH 3.5; 3% malt extract agar, pH 5.5; and plate count agar, pH 7.0 were used to enumerate CFUs produced in pour plates. (Preliminary studies indicated that pour and spread plates gave similar results). Plate count agar became the medium of choice after it was found to yield somewhat higher recoveries. Freshly prepared rose bengal dye was added to the media to give a final concentration of 8.4 mg/L. Its function was to reduce spreading of the mold colonies. An incubation of 4-5 d at 30°C yielded maximal counts.