Proof of concept: could snake venoms be a potential source of bioactive compounds for control of mould growth and mycotoxin production.

The objective was to screen 10 snake venoms for their efficacy to control growth and mycotoxin production by important mycotoxigenic fungi including Aspergillus flavus, Aspergillus westerdijkiae, Penicillium verrucosum, Fusarium graminearum and F. langsethiae. The Bioscreen C rapid assay system was used. The venoms from the Viperidae snake family delayed growth of some of the test fungi, especially F. graminearum and F. langsethiae and sometimes A. flavus. Some were also able to reduce mycotoxin production. The two most potent crude snake venoms (Naja nigricollis and N. siamensis; 41 and 43 fractions, respectively) were further fractionated and 83/84 of these fractions were able to reduce mycotoxin production by >90% in two of the mycotoxigenic fungi examined. This study suggests that there may be significant potential for the identification of novel fungistatic/fungicidal bioactive compounds as preservatives of raw and processed food commodities post-harvest from such snake venoms.

Impact of climate change environmental conditions on the resilience of different formulations of the biocontrol agent Candida sake CPA-1 on grapes.

Biocontrol agents have become components of integrated crop protection systems for controlling economically important fungal pathogens. Candida sake CPA-1 is a biocontrol agent of fungal pathogens of fruits, both pre- and post-harvest. While the efficacy of different formulations have been examined previously, few studies have considered the resilience of different formulations under changing climatic conditions of elevated temperature, drought stress and increased atmospheric CO2 . This study examined the effect of (a) temperature × RH × elevated CO2 (400 vs 1000 ppm) on the temporal establishment and viability of two dry and one liquid C. sake CPA-1 formulations on grape berry surfaces; (b) temperature stress (25 vs 35°C); and (c) elevated CO2 levels. Results indicated that temperature, RH and CO2 concentration influenced the establishment and viability of the formulations but there was no significant difference between formulations. For the combined three-component factors, increased temperature (35°C) and lower RH (40%) reduced the viable populations on grapes. The interaction with elevated CO2 improved the establishment of viable populations of the formulations tested. Viable populations greater than Log 4 CFUs per g were recovered from the grape surfaces suggesting that these had conserved resilience for control of Botrytis rot in grapes. SIGNIFICANCE AND IMPACT OF THE STUDY: The interaction between environmental factors that are expected to occur in response to climate change (CC) will have a significant impact on food security and availability. Little information exists on how elevated temperature, drought stress and increased CO2 will have on the efficacy of biocontrol agents. The impact of these factors on the viability of different formulations of the biocontrol yeast Candida sake on the surface of grapes berries was evaluated for the first time. Such knowledge is critical for projecting the efficacy of biocontrol under climate change conditions and to identify formulations that have the necessary resilience to perform under CC conditions.

Efficacy of different caffeine concentrations on growth and ochratoxin A production by Aspergillus species.

UNLABELLED: The objective of this study was to evaluate the effect of different caffeine concentrations (0-4%) on (i) lag phase prior to growth, (ii) growth rates and (iii) ochratoxin A (OTA) production by strains from the Aspergillus section Circumdati and Aspergillus section Nigri groups, isolated from coffee, when grown on a conducive medium at 0·98 water activity and 30°C. The lag phases prior to growth increased with caffeine concentration. A strain of Aspergillus niger and Aspergillus carbonarius were the most sensitive to caffeine with growth being inhibited by <1% caffeine. For strains of Aspergillus westerdijkiae, Aspergillus ochraceus and Aspergillus steynii, although growth was inhibited significantly, some growth (10-15% of controls) occurred in 4% caffeine. OTA production was significantly inhibited by only 0·5% caffeine for strains of A. westerdijkiae, A. niger and A. carbonarius. For A. steynii at least 1·5% caffeine was required to inhibit OTA production. In contrast, for the strain of A. ochraceus there was a stimulation of OTA at 3% with a reduction at 4% caffeine. These results are discussed in the context of the different concentrations of caffeine found in Arabica and Robusta coffee and the development of minimization strategies. SIGNIFICANCE AND IMPACT OF THE STUDY: Arabic (0·6%) and Robusta coffee (4%) have significantly different amounts of endogenous caffeine. The growth of six ochratoxigenic fungi which contaminate coffee with ochratoxin A (OTA) had differential tolerance/sensitivity to concentrations of caffeine in vitro in this range. However, low concentrations of caffeine (<0·5%) was inhibitory to OTA production. These results are discussed in the context of the potential for using such information for the design of minimization strategies to control mycotoxin production in such products.

Temperature and water activity effects on production of T-2 and HT-2 by Fusarium langsethiae strains from north European countries.

This study has examined the effect of ecophysiological factors, water activity (a(w), 0.995-0.90) and temperature (10-37 °C), on the T-2 and HT-2 toxins production by Fusarium langsethiae. Two dimensional profiles for optimum and marginal conditions have been built for two strains from each of four northern European countries (UK, Norway, Sweden, Finland) on an oat-based medium. This showed that the optimum a(w) and temperature conditions for T-2 + HT-2 production was between 0.98-0.995, and 20-30 °C respectively. Kruskal-Wallis analysis of ranks showed a statistically significant differences between the different a(w) levels examined (P < 0.001) but no significant effect of the temperatures examined. The ratio of HT-2/T-2 was investigated and non-uniform distribution of HT-2 toxin was found under different ecological conditions. No statistically significant differences were found for the mean toxin production between strains from the different countries. Intra-strain differences in toxin production was only found for those from Finland (P-value = 0.0247). The growth/no growth and toxin/no toxin conditions in relation to a(w) x temperature have been constructed for the first time. This knowledge will be useful in developing prevention strategies to minimise T-2 and HT-2 toxin contamination by strains of F. langsethiae on important small grain cereals.

Fungal diversity and metabolomic profiles in GM and isogenic non-GM maize cultivars from Brazil.

There is little knowledge of the microbial diversity, mycotoxins and associated secondary metabolites in GM maize and isogenic non-GM cultivars (cvs). This study has quantified the microbial populations and dominant fungal genera in 6 cvs of each type representative of herbicide, pesticide or stacked resistance to both. The predominant mycotoxins and targeted metabolomics profiles were also compared between the two sets of cvs. This showed that the overall fungal populations were 8.8 CFUs g-1 maize. The dominant genera, isolated from maize samples, whether surface-sterilised or not, in all maize cvs were Fusarium, followed by Penicillium, Aspergillus and occasionally Cladosporium and Alternaria. The analysis of the targeted metabolomics showed that approx. 29 different metabolites were detected. These were dominated by fumonisins and minor Penicillium spp. metabolites (questiomycin A and rugulovasine A). Interestingly, the range and number of mycotoxins present in the GM cvs were significantly lower than in the non-GM maize samples. This suggests that while the fungal diversity of the two types of maize appeared to be very similar, the major contaminant mycotoxins and range of toxic secondary metabolites were much lower in the GM cvs.

Temporal monitoring of the nor-1 (aflD) gene of Aspergillus flavus in relation to aflatoxin B1 production during storage of peanuts under different water activity levels.

AIMS: A relative quantification system (RQ-PCR) was used to monitor the correlations between the activity of the nor-1 (=aflD) gene of Aspergillus flavus using real-time PCR in relation to phenotypic aflatoxin B(1) (AFB(1) ) production and populations of A. flavus in stored peanuts at three water activity levels (a(w) , 0·95, 0·90 and 0·85) for 6 weeks. METHODS AND RESULTS: Real-time PCR was used to amplify the nor-1 gene (target gene), and benA56 (ß-tubulin gene) used as a control gene. Expression of three structural genes, nor-1 (=aflD), ver-1 (=aflM), and omtA (=aflP), and the regulatory gene aflR of the aflatoxin biosynthetic pathway were also assayed. There were significant differences between nor-1 gene expression at the three a(w) levels; higher expression at 0·90 a(w) in weeks 1-3, when compared to 0·95. In contrast, in the driest treatment (0·85 a(w) ) none or very low nor-1 expression occurred. The populations of A. flavus colony-forming units (CFUs g(-1) ) increased over time with the highest at 0·95 a(w) . Highest AFB(1) production was at 0·90 and 0·95 a(w) from weeks 3-6. A(w) had a significant effect on aflR transcription at 0·95 a(w) over the 6-week period, while at 0·90 a(w) , only in the last 2 weeks. CONCLUSIONS: Correlations between different factors showed that log AFB(1) × log CFUs, log AFB(1) × a(w) , and log CFUs × a(w) were statistically significant, while log CFUs × RQ-PCR and RQ-PCR × a(w) were not. The AflR gene may not have an important role in the regulation of nor-1 expression in food matrices (e.g. peanuts). SIGNIFICANCE AND IMPACT OF THE STUDY: Determination of correlations between nor-1 expression and aflatoxin production by A. flavus in raw peanuts under different a(w) levels could be helpful to predict potential risk of aflatoxin production during storage of this hygroscopic food product and minimize contamination with the AFB(1) .

Efficacy of metabolites of a Streptomyces strain (AS1) to control growth and mycotoxin production by Penicillium verrucosum, Fusarium verticillioides and Aspergillus fumigatus in culture.

The objectives of this study were to determine the efficacy of metabolites of a Streptomyces strain AS1 on (a) spore germination, (b) mycelial growth, (c) control of mycotoxins produced by Penicillium verrucosum (ochratoxin A, OTA), Fusarium verticillioides (fumonisins, FUMs) and Aspergillus fumigatus (gliotoxin) and (d) identify the predominant metabolites involved in control. Initial screening showed that the Streptomyces AS1 strain was able to inhibit the mycelial growth of the three species at a distance, due to the release of secondary metabolites. A macroscopic screening system showed that the overall Index of Dominance against all three toxigenic fungi was inhibition at a distance. Subsequent studies showed that the metabolite mixture from the Streptomyces AS1 strain was very effective at inhibiting conidial germination of P. verrucosum, but less so against conidia of A. fumigatus and F. verticillioides. The efficacy was confirmed in studies on a conducive semi-solid YES medium in BioScreen C assays. Using the BioScreen C and the criteria of Time to Detection (TTD) at an OD = 0.1 showed good efficacy against P. verrucosum when treated with the Streptomyces AS1 extract at 0.95 and 0.99 water activity (aw) when compared to the other two species tested, indicating good efficacy. The effective dose for 50% control of growth (ED50) at 0.95 and 0.99 aw were approx. 0.005 ng/ml and 0.15 µg/ml, respectively, with the minimum inhibitory concentration (MIC) at both aw levels requiring > 40 µg/ml. In addition, OTA production was completely inhibited by 2.5 µg/ml AS1 extract at both aw levels in the in vitro assays. Ten metabolites were identified with four of these being predominant in concentrations > 2 µg/g dry weight biomass. These were identified as valinomycin, cyclo(L-Pro-L-Tyr), cyclo(L-Pro-L-Val) and brevianamide F.

Carbon dioxide production as an indicator of Aspergillus flavus colonisation and aflatoxins/cyclopiazonic acid contamination in shelled peanuts stored under different interacting abiotic factors.

Aspergillus flavus is the main xerophylic species colonising stored peanuts resulting in contamination with aflatoxins (AFs) and cyclopiazonic acid (CPA). This study evaluated the relationship between storage of shelled peanuts under interacting abiotic conditions on (a) temporal respiration (R) and cumulative CO2 production, (b) dry matter losses (DMLs) and (c) aflatoxin B1 (AFB1) and CPA accumulation. Both naturally contaminated peanuts and those inoculated with A. flavus were stored for 7-days under different water activities (aw; 0.77-0.95) and temperatures (20-35°C). There was an increase in the temporal CO2 production rates in wetter and warmer conditions, with the highest respiration at 0.95 aw + A. flavus inoculum at 30°C (2474 mg CO2kg-1h-1). The DMLs were modelled to produce contour maps of the environmental conditions resulting in maximum/minimum losses. Maximum mycotoxin contamination was always at 0.95 aw although optimal temperatures were 25-30°C for AFs and 30-35°C for CPA. These results showed a correlation between CO2 production and mycotoxin accumulation. They also provide valuable information for the creation of a database focused on the development of a post-harvest decision support system to determine the relative risks of contamination with these mycotoxins in stored shelled peanuts.

Effect of temperature and water activity on growth and ochratoxin A production boundaries of two Aspergillus carbonarius isolates on a simulated grape juice medium.

AIMS: To develop and validate a logistic regression model to predict the growth and ochratoxin A (OTA) production boundaries of two Aspergillus carbonarius isolates on a synthetic grape juice medium as a function of temperature and water activity (a(w)). METHODS AND RESULTS: A full factorial design was followed between the factors considered. The a(w) levels assayed were 0.850, 0.880, 0.900, 0.920, 0.940, 0.960, 0.980 and the incubation temperatures were 10, 15, 20, 25, 30, 35 and 40 degrees C. Growth and OTA production responses were evaluated for a period of 25 days. Regarding growth boundaries, the degree of agreement between predictions and observations was >99% concordant for both isolates. The erroneously predicted growth cases were 3.4-4.1% false-positives and 0.7-1.4% false-negatives. No growth was observed at 10 degrees C and 40 degrees C for all a(w) levels assayed, with the exception of 0.980 a(w)/40 degrees C, where weak growth was observed. Similarly, OTA production was correctly predicted with a concordance rate >98% for the two isolates with 0.7-1.4% accounting for false-positives and 2.0-2.7% false-negatives. No OTA production was detected at 10 degrees C or 40 degrees C regardless of a(w), and at 0.850 a(w) at all incubation temperatures. With respect to time, the OTA production boundary shifted to lower temperatures (15-20 degrees C) as opposed to the growth boundary that shifted to higher temperature levels (25-30 degrees C). Using two literature datasets for growth and OTA production of A. carbonarius on the same growth medium, the logistic model gave one false-positive and three false-negative predictions out of 68 growth cases and 13 false-positive predictions out of 45 OTA production cases. CONCLUSIONS: The results of this study suggest that the logistic regression model can be successfully used to predict growth and OTA production interfaces for A. carbonarius in relation to temperature and a(w). SIGNIFICANCE AND IMPACT OF THE STUDY: The proposed modelling approach helps the understanding of fungal-food ecosystem relations and it could be employed in risk analysis implementation plans to predict the risk of contamination of grapes and grape products by A. carbonarius.

Influence of storage environment on maize grain: CO2 production, dry matter losses and aflatoxins contamination.

Poor storage of cereals, such as maize can lead to both nutritional losses and mycotoxin contamination. The aim of this study was to examine the respiration of maize either naturally contaminated or inoculated with Aspergillus flavus to examine whether this might be an early and sensitive indicator of aflatoxin (AF) contamination and relative storability risk. We thus examined the relationship between different interacting storage environmental conditions (0.80-0.99 water activity (aw) and 15-35°C) in naturally contaminated and irradiated maize grain + A. flavus on relative respiration rates (R), dry matter losses (DMLs) and aflatoxin B1 and B2 (AFB1-B2) contamination. Temporal respiration and total CO2 production were analysed by GC-TCD, and results used to calculate the DMLs due to colonisation. AFs contamination was quantified at the end of the storage period by HPLC MS/MS. The highest respiration rates occurred at 0.95 aw and 30-35°C representing between 0.5% and 18% DMLs. Optimum AFs contamination was at the same aw at 30°C. Highest AFs contamination occurred in maize colonised only by A. flavus. A significant positive correlation between % DMLs and AFB1 contamination was obtained (r = 0.866, p < 0.001) in the irradiated maize treatments inoculated with A. flavus. In naturally contaminated maize + A. flavus inoculum loss of only 0.56% DML resulted in AFB1 contamination levels exceeding the EU legislative limits for food. This suggests that there is a very low threshold tolerance during storage of maize to minimise AFB1 contamination. This data can be used to develop models that can be effectively used in enhancing management for storage of maize to minimise risks of mycotoxin contamination.

Impact of mild heat treatments on induction of thermotolerance in the biocontrol yeast Candida sake CPA-1 and viability after spray-drying.

AIMS: The objective of this study was to examine the induction of thermotolerance in the biocontrol agent Candida sake CPA-1 cells by mild heat treatments to enhanced survival of formulations using spray-drying. The possible role of heat-shock proteins (HSPs) biosynthesis in induced thermotolerance and the role of sugars and sugar alcohols were also determined. METHODS AND RESULTS: Studies were conducted on C. sake cells grown in molasses medium and exposed to mild temperatures of 30 and 33 degrees C during mid- (16 h), late-exponential (24 h), early- (30 h) and mid-stationary (36 h) growth phases. The effect on viability was determined both before and after spray-drying. Cycloheximide and chloramphenicol were used to examine the role of HSPs and HPLC was used to analyse the accumulation of sugar and sugar alcohols. The results indicate that both temperatures induced thermotolerance in cells of C. sake. Mild heat-adapted cells at 33 degrees C in the early- or mid-stationary phases had survival values after spray-drying significantly higher (P

Studies on Aspergillus section Flavi isolated from maize in northern Italy.

In 2003, for the first time in Italy, significant problems arose with colonization and contamination of maize destined for animal feed with Aspergillus section Flavi and aflatoxins (AFs). This resulted in milk and derived products being contaminated with AFM(1) at levels above the legislative limit. There was little knowledge and experience of this problem in Italy. The objectives of this research were thus to study the populations of Aspergillus section Flavi in six northern Italian regions and obtain information on the relative role of the key species, ability to produce sclerotia, production of the main toxic secondary metabolites, aflatoxins and cyclopiazonic acid, and tolerance of key environmental parameters. A total of 70 strains were isolated and they included the toxigenic species A. flavus and A. parasiticus. A. flavus was dominant in the populations studied, representing 93% of the strains. Seventy percent of strains of Aspergillus section Flavi produced AFs, with 50% of strains also producing cyclopiazonic acid. Sixty-two percent of A. flavus strains and 80% of A. parasiticus were able to produce sclerotia at 30 degrees C. Using 5/2 agar, only 1 strain developed S sclerotia and 19 L sclerotia. With regard to ecological studies, growth of Aspergillus section Flavi was optimal at between 25 and 30 degrees C, while AFB(1) production was optimal at 25 degrees C. Regarding water availability (water activity, a(w)), 0.99 a(w) was optimal for both growth and AFs production, while the only aflatoxin produced in the driest condition tested (0.83 a(w)) was AFB(1). This information will be very useful in identifying regions at risk in northern Italy by linking climatic regional information to levels of fungal contamination present and potential for aflatoxin production in maize destined for animal feed. This would be beneficial as part of a prevention strategy for minimising AFs in this product.

European research on ochratoxin A in grapes and wine.

European wine production represents about 70% of world production and thus is an important export commodity. Ochratoxin A (OTA) was first detected as a wine contaminant in 1996 and the role of Aspergillus section Nigri and A. carbonarius in OTA production discovered in Europe in 1999. Subsequently Europe-wide surveys have shown that A. carbonarius is predominantly responsible for OTA contamination of grapes, wine and vine fruits. Analyses of wine samples throughout Europe have shown that there is a gradient in OTA concentration with a decrease from red, to rose and to white wines. The latitude of production is an important factor in determining risk from OTA contamination. Some geographic regions in Southern Europe are more prone to contamination with the toxigenic species and OTA. Ochratoxin A has also been found in much higher concentrations (max. 53 mug/kg) in dried vine fruit than in wine suggesting that A. carbonarius can dominate the drying vine fruit ecosystem. There is a significant lack of knowledge in Europe on conducive climatic conditions preharvest and their relationship with levels of risk from OTA contamination in grapes and their fate in wine production. This needs to be integrated with cultivation system to maximise the prevention of OTA entering this food chain.

Mapping of Aspergillus Section Nigri in Southern Europe and Israel based on geostatistical analysis.

Geostatistical analysis was applied to the incidence of Aspergillus Section Nigri and A. carbonarius in Southern Europe and Israel for the 3-year period 2001-2003 to facilitate identification of regions of high risk from contamination with these fungi and production of ochratoxin. The highest incidence of black aspergilli was normally observed at harvesting. At this grape growth stage, spatial variability of black aspergilli was significantly related to latitude and longitude, showing a positive West-East and North-South gradient. Predictive maps of infected berries incidence were drawn and showed the same trend in the 3 years, but incidence was highest in 2003, followed by 2001 and 2002. The highest incidence was always observed in Israel, Greece and Southern France, associated with the highest incidence of A. carbonarius. Southern Spain and Southern Italy also had relevant incidence of black aspergilli. The thermo-wetness maps for the 3 years showed a trend similar to the incidence of black aspergilli. The coldest and wettest year was 2002, while 2003 was the hottest and driest, particularly during August, with Israel being the hottest and driest country, followed by Greece and Southern Italy. This indicates that meteorological conditions can contribute to explain spatial distribution variation of black aspergilli within the Mediterranean basin.

An intelligent rapid odour recognition model in discrimination of Helicobacter pylori and other gastroesophageal isolates in vitro.

Two series of experiments are reported which result in the discrimination between Helicobacter pylori and other bacterial gastroesophageal isolates using a newly developed odour generating system, an electronic nose and a hybrid intelligent odour recognition system. In the first series of experiments, after 5 h of growth (37 degrees C), 53 volatile 'sniffs' were collected over the headspace of complex broth cultures of the following clinical isolates: Staphylococcus aureus, Klebsiella sp., H. pylori, Enterococcus faecalis (10(7) ml(-1)), Mixed infection (Proteus mirabilis, Escherichia coli, and E. faecalis 3 x 10(6) ml each) and sterile cultures. Fifty-six normalised variables were extracted from 14 conductive polymer sensor responses and analysed by a 3-layer back propagation neural network (NN). The NN prediction rate achieved was 98% and the test data (37.7% of all data) was recognised correctly. Successful clustering of bacterial classes was also achieved by discriminant analysis (DA) of a normalised subset of sensor data. Cross-validation identified correctly seven 'unknown' samples. In the second series of experiments after 150 min of microaerobic growth at 37 degrees C, 24 volatile samples were collected over the headspace of H. pylori cultures in enriched (HPP) and normal (HP) media and 11 samples over sterile (N) cultures. Forty-eight sensor parameters were extracted from 12 sensor responses and analysed by a 3-layer NN previously optimised by a genetic algorithm (GA). GA-NN analysis achieved a 94% prediction rate of 'unknown' data. Additionally the 'genetically' selected 16 input neurones were used to perform DA-cross validation that showed a clear clustering of three groups and reclassified correctly nine 'sniffs'. It is concluded that the most important factors that govern the performance of an intelligent bacterial odour detection system are: (a) an odour generation mechanism, (b) a rapid odour delivery system similar to the mammalian olfactory system, (c) a gas sensor array of high reproducibility and (d) a hybrid intelligent model (expert system) which will enable the parallel use of GA-NNs and multivariate techniques.

Ecological determinants of mould growth in stored grain.

Grain entering store carries a microflora of 'field' and 'storage' fungi. Field fungi require readily available water and therefore seldom develop in store. By contrast, storage fungi, especially Aspergillus spp., are able to grow at low water activities (aw, 0.70-0.75) enabling them to initiate grain spoilage. The ability of storage fungi to germinate, grow and sporulate in stored grain is dependent on the availability of water in the substrate, temperature and the intergranular gas composition. These factors may interact to have a profound influence on the initiation of spoilage of stored grain by fungi. An understanding of the ecological determinants of mould growth may help to develop improved and safer methods of grain storage.

Impact of environment and interspecific interactions between spoilage fungi and Aspergillus ochraceus on growth and ochratoxin production in maize grain.

Using layers of irradiated but still fertile maize grain, the effects of water activity (0.995, 0.95 a(w)) and temperature (18, 30 degrees C) on interspecific interactions between Aspergillus ochraceus and five other spoilage fungi were examined. Asp. ochraceus was not competitive against Asp. flavus, Asp. niger, or Alternaria alternata at 18 degrees C when water was freely available (0.995 a(w)), while at 0.95 a(w) it was dominant against Asp. candidus, Asp. flavus and Alt. alternata. At 30 degrees C and 0.995 a(w), Asp. ochraceus was dominated by other fungi, except Alt. alternata, and was mutually antagonistic to Asp. candidus and Eurotium amstelodami. However, at 30 degreees C and 0.95 a(w), it was competitive against Asp. candidus and Alt. alternata, but not against the other species examined. The overall Index of Dominance showed that Asp. ochraceus was not competitive under the conditions examined here. At 18 degrees C ochratoxin production by Asp. ochraceus was inhibited significantly by Asp. candidus (0.995 and 0.95 a(w)) and Asp. niger (0.995 a(w)). When grown on maize grain at 30 degrees C, ochratoxin production by Asp. ochraceus was significantly inhibited by other spoilage fungi when both were grown on maize grain, especially by Asp. niger and E. amstelodami (0.995 a(w)) and Asp. flavus at 0.95 a(w). These results suggest that, to a large extent, A. ochraceus is not as competitive as some other spoilage fungi in primary resource capture on maize grain at a(w) of 0.95 or above, although it may modify resource quality and influence secondary colonisation by other species under the conditions tested.

Lipolytic activity and degradation of rapeseed oil and rapeseed by spoilage fungi.

Aspergillus, Eurotium and Penicillium spp. from rapeseed were able to grow and produce lipases over a range of water activities (aw) at both 15 and 25 degrees C on tributyrin agar. The ability to produce lipases was not directly related to growth rate. The clearing zone: growth rate ratios gave lipase indices which varied markedly between test fungi and with aw. The fungi with the highest indices were Aspergillus candidus and Aspergillus versicolor and Penicillium expansum and Penicillium hordei. The Aspergillus spp. generally grew faster on a 1% crude rapeseed oil at 25 degrees C and 0.995 and 0.95 aw than on the tributyrin agar. P. hordei degraded the rapeseed oil more rapidly than Eurotium amstelodami or Penicillium aurantiogriseum, with optimum activity at 0.98 aw. E. amstelodami was the least effective at degrading the rapeseed oil under all aw conditions. The lipase indices were compared with the ability of the test fungi to degrade irradiated rapeseed. Dry matter loss over 4-week periods were greater at 25 than 15 degrees C, regardless of aw. At 15 degrees C, Penicillium spp. caused significantly greater dry matter losses than Aspergillus spp. A. niger degraded rapeseed faster than other Aspergillus spp. tested at 0.98 aw, but at 0.95 and 0.90 aw there was little difference between species.

Effect of water activity on hydrolytic enzyme production by Fusarium moniliforme and Fusarium proliferatum during colonisation of maize.

The effect of different water availabilities (water activity, aw; 0.98-0.93) and time (up to 15 days) on the production of seven hydrolytic enzymes by strains of F. moniliforme and F. proliferatum during early colonisation of gamma-irradiated living maize grain were examined in this study. Both the total activity (micromol 4-nitrophenol min(-1) g(-1) maize) and specific activity (nmol 4-nitrophenol min(-1) microg(-1) protein) were quantified using chromogenic p-nitrophenyl substrates. The dominant three enzymes produced by the fungi on whole colonised maize kernels were alpha-D-galactosidase, beta-D-glucosidase, and N-acetyl-beta-D-glucosaminidase. The other four enzymes were all produced in much lower total amounts and in terms of specific activity (beta-D-fucosidase, alpha-D-mannosidase, beta-D-xylosidase and N-acetyl-alpha-D-glucosaminidase), similar to that in uncolonised control maize grain. There were significant increases in the total production of the three predominant enzymes between 3-15 days colonisation, and between 3-6 days in terms of specific activity when compared to untreated controls. The total and specific activity of the alpha-D-galactosidase, beta-D-glucosidase and N-acetyl-beta-D-glucosaminidase, were maximum at 0.98 aw with significantly less being produced at 0.95 and 0.93 aw, with the exception of the total activity of alpha-D-galactosidase which was similar at both 0.95 and 0.93 aw. Single factors (time, aw, and inoculation treatment), two- and three- way interactions were all statistically significant for the three dominant enzymes produced except for specific activity of beta-D-glucosidase (two and three-way interactions) and for total activity of alpha-D-galactosidase in the time x aw treatment. This study suggests that these hydrolytic enzymes may play an important role in enabling these important fumonisin-producing Fusarium spp. to rapidly infect living maize grain over a wide aw range.