Ecophysiology and water relations of growth and ochratoxin A production by Penicillium verrucosum adn Aspergillus westerdijkiae, impacts of climate change and control using preservatives.

Abstract

Penicillium verrucosum and Aspergillus westerdijkiae contaminate cereal grains and coffee beans with ochratoxin A (OTA) for which legislative limits exist. These fungi reside in soil and contaminate grain during harvesting and post-harvest storage, and post-fermentation during drying of coffee beans. There is a lack of information on the impacts of environmental conditions which influence the inoculum potential in soil and on cereal grain and coffee beans and potential control of OTA during post-harvest storage phases. The objectives of this project were to: (a) examine the water and temperature relations of strains of P. verrucosum (two strains) and A. westerdijkiae (three strains) in relation to growth and OTA production, (b) compare the sensitivity and tolerance of one strain of each species to solute (ionic and non-ionic) and matric stress on growth kinetics, expression of key genes in the OTA cluster (otapks, otanrps) and phenotypic OTA production for the first time, (c) examine the impact of three way interacting climate change related abiotic environmental factors (water activity, temperature, and CO₂ levels) on growth, gene expression and OTA production by strains of the two species, and (d) screen a range of potential preservatives in vitro control of growth and OTA production for in situ control of contamination of grain and coffee under different aw and temperature conditions. The ecological studies revealed that both strains of P. verrucosum (OTA11; straw21) could grow over a wide range of aw levels and temperatures. However, under drier conditions (0.90 aw) growth was much slower. For OTA production, optimum production was at 25°C and 0.98 aw for strain OTA11 when compared to the other strain on a conducive YES medium. On wheat-based media, both strains were able to grow efficiently over a range of aw levels (0.98, 0.95 and 0.90 aw) at 15, 20 and 25°C. On wheat-based matrices, the OTA production was much lower than on the conducive YES medium, regardless of aw and temperature for both strains after 10 days incubation. However, after 20 days, the strain Straw21, produced higher amounts of OTA under water stress condition (0.90 aw) at 25°C. For A. westerdijkiae strains growth occurred over a wide range of aw levels and different temperatures on the conducive YES media. However, the strain A. westerdijkiae CECT produced the highest amount of OTA at 0.98 aw and 25-30°C. Comparison of growth under solute and matric stress showed that the growth of P. verrucosum strain OTA11 is more tolerant of matric potential stress than Straw21 over a wide range of water potentials at 25°C. The strain OTA11 grew well under all non-ionic solute potential stress and under matric stress. However, under ionic solute stress no growth occurred under extreme water stress of -19.6 MPa (=0.86 aw). Upon examination of the expression pattern of the otapks gene under such stress conditions, there was an increase in the gene expression in the non-ionic (glycerol) solute stress-imposed treatments when compared with ionic solute (NaCl) and matric stress treatments. This suggests that this is a key gene involved in OTA biosynthesis. There were some parallels with the phenotypic OTA production. For A. westerdijkiae species, both tested strains (CECT and CCT) grew well under matric stress at all stress levels imposed (-1.4—19.6 MPa = 0.99-0.86 aw). As for P. verrcuosum, this strain was more sensitive to ionic solute stress imposed with NaCl with growth inhibited at -19.6 MPa (=0.86 aw). For this species, the gene expression of otapks was significantly increased under moderate stress conditions -9.8 MPa (=0.95 aw) modified with the non-ionic solute (glycerol). However, under matric stress this expression was significantly reduced when compared to solute stress. For OTA production, this was increased at -9.8 MPa (=0.95 aw) under non-ionic solute stress when compared to the other treatments. Overall, the impact of climate change related abiotic factors, especially elevated CO₂ levels (400 vs 1000 ppm CO₂) had no significant effect on the growth of P. verrucosum when compared with existing conditions under matric stress. However, under non-ionic stress (glycerol imposed), no growth was reported at -2.8 MPa (=0.95 aw) at 1000 ppm and 30°C. Overall, the growth pattern in non-ionic solute stress was lower under elevated levels of CO₂ than in matric stress conditions when compared with existing conditions. For the otapks gene, expression was increased under elevated CO₂ levels in matric stress treatments when compared to existing conditions. This pattern was paralleled with production of OTA under these conditions. With regard to A. westerdijkiae, surprisingly no growth occurred at 37°C in all the conditions tested. However, at 30°C, the elevated levels of CO₂ had no significant impact on growth under matric and non-ionic stress when compared with existing CO₂levels. For the otapks gene expression, this increased in matric imposed water stress in all conditions examined. However, there was no gene expression in non-ionic stress conditions, and this paralleled the OTA production pattern. Initial screening of six potential preservatives showed that for both growth and OTA control by one strain of each species SM, TCA and PP were the most effective compounds. They inhibited growth of P. verrucosum at 250 ppm on wheat-based matrices. While, for FE, it was the least effective treatment as ED50 and ED90 values show that 1000 mg/l and 2700 mg/l are required for controlling the growth rate at both water stress levels (0.95 and 0.95 aw) respectively and for the MIC for toxin production. However, on stored wheat grains, some growth and OTA was produced in the SM and TCA treatments at 250 ppm treatment. For A. westerdijkiae, the most effective compounds inhibiting growth were at 500 and 1000 ppm of SM, TCA and PP at 0.95 and 0.98 aw on coffee-based media. With PP, no toxin was produced at 100 ppm at both water stress levels although some growth occurred. Also, FE was the least effective treatment with ED50 and ED90 values of 1000 and 1530 mg/l respectively at both water stress levels. In stored coffee beans, the results were different with some growth found at 1000 ppm in treatments of TCA and SM at both aw levels. In addition, high amounts of OTA were produced in the 1000 ppm treated and stored coffee beans at both aw levels. Overall, in vitro efficacy was not an accurate guide to in situ efficacy, especially in relation to toxin control in both stored wheat and coffee beans under different aw levels.