Comparative study for growth and sporulation of some mycotoxigenic fungi in relation to water activity effects

This study examined the effect of water activity (0.85-0.995 aw) on growth rate and asexual spore production for four mycotoxigenic strains (Aspergillus flavus, Aspergillus ochraceus, Aspergillus carbonarius and Penicillium verrucosum) on Malt Extract Agar (MEA). The water activity levels of MEA media were modified ionically (NaCl) and non-ionically (glycerol). Results showed that the optimum aw for growth was at 0.98-0.995 for all species using both solutes. However, when water stress was inflicted, there was a slower growth for all species. The limit for growth of the strains was at 0.85-0.9 aw, there was no growth at 0.9 aw for A. carbonarius using NaCl solute and 0.85 aw for A. carbonarius and A. flavus using glycerol solute. A. ochraceus and P. verrucosum had a higher tolerance to lower water activity than A. carbonarius and A. flavus when modified with NaCl. There were significant differences in sporulation between species on glycerol and NaCl-amended media. The optimum conditions for production of asexual spores is often very different from that for growth. Little amount of conidial spore occurred at 0.93-0.95 aw modified with NaCl in cultures of A. carbonarius and P. verrucosum but high amounts were produced by A. ochraceus and A. flavus. Optimum water activity for spore production was 0.995 aw for A. carbonarius, 0.98 aw for P. verrucosum, 0.95 aw for A. flavus and 0.85 aw for A. ochraceus on modified media with glycerol. This is the first detailed study to examine the similarities and differences in growth and sporulation in response to the change of water activity level of important mycotoxigenic species. This study can help in understanding why these species are varied in mycotoxin production, so the results obtained in this study may be useful for application in systems of food safety management.


INTRODUCTION
Food and animal feed can be contaminated with mycotoxigenic filamentous fungi by producing one or more potential mycotoxins.Globally, 25 to 50% of crops that are produced for food and feed are vulnerable for E-mail: ahmed_alhadi2000@yahoo.com.Tel: 00966540589706.
Author(s) agree that this article remains permanently open access under the terms of the Creative Commons Attribution License 4.0 International License contamination with mycotoxins.The percentage could be more in tropical areas and it is documented that up to 80% of the crops are reported to contain major amounts of mycotoxins (Konietzny and Greiner, 2003).Food and feed can be easily contaminated with aflatoxigenic and ochratoxigenic fungi, especially in warm climates.Aspergillus ochraceus and black aspergilli, such as Aspergillus niger and Aspergillus carbonarius, are ochratoxin producing species most frequently found in warm and tropical regions of the world, while in temperate climates ochratoxin (OTA) is principally produced by Penicillium verrucosum (Pitt and Hocking, 1997;Pardo et al., 2005).Exposure to OTA has been linked to the progressive kidney disease known as Balkan endemic nephropathy (BEN) and to the development of urinary tract tumours in humans (Plestina, 1996).Aspergillus flavus is the main producer of aflatoxins (AFs), which include the most potent natural carcinogen known (JECFA, 1997).Some isolates of this species are able to produce other mycotoxins, particularly cyclopiazonic acid (CPA), which is toxic to a variety of animals and has been implicated in human poisoning (Dorner et al., 1983;Rao and Husain, 1985).
Spoliage of agricultural products occurs as a result of infection by mycotoxigenic fungi under suitable environmental conditions in the field and may occur at various stages of food chain, for example, pre-harvesting, harvesting, drying, and storage (Sinha, 1995).Depending on the environmental conditions and the specific water activity (a w ), the specific fungal species will grow and produce toxins (Sanchis and Magan, 2004).The role of water activity in determining growth, evolution and adhesion degree of moulds on nutrient substrates is well recognized ( Bouras et al., 2009).When the fungal growth is not restricted by environmental factors such as pH or temperature, chemical reactions, enzymatic changes, and microbial growth may occur readily in foods with high water contents.Fungal growth and mycotoxin production in foods and feeds depend on the effect of biotic and abiotic factors including water activity and temperature (Astoreca et al., 2012).Water activity is more useful parameter than water content as it reflects the availability of water for metabolic processes (Sanchis and Magan, 2004).
It has previously been shown that a w is one of the important criteria for understanding the ecology of spoilage fungi, especially mycotoxigenic species.A significant amount of information is now available on the growth and mycotoxin profiles for many of these species including ochratoxigenic ones and aflatoxigenic onces (Garcia et al., 2011;Astoreca et al., 2012).It has been examined the impact that a w x temperature factors on growth and mycotoxin production by a wide range of mycotoxigenic fungi (Sanchis and Magan, 2004;Magan and Aldred, 2007).They reported that the range of a w x temperature for mycotoxin production is generally more restrictive than those for growth.In contrast to Penicillium verrucosum which can grow and produce ochratoxin A under a very similar range of a w x temperature conditions (Cairns-Fuller et al., 2005).Generally, most moulds do not grow at a water activity (a w ) of 0.8.However, practically no information is available on the similarities and differences between these species, which can produce ochratoxins or aflatoxins.Thus, the aim of this study was to compare the in vitro effect of the key ecological parameter of a w modified by glycerol and NaCl on growth rate, and a sexual spore production of four mycotoxigenic strains.

Fungal strain
In this study, four mycotoxigenic fungal strains (A.flavus 2092, A. ochraceus 14027, A. carbonarius IT 1102 and P. verrucosum 593) were tested.The strains were kindly supplied by Prof Naresh Magan (Applied Mycology Group, Cranfield Health, Cranfield University, Bedford, UK).The strains were sub-cultured on Malt Extract Agar (20 g malt extract, 2 g peptone, 15 g agar per liter) for 7 days at 25°C in the dark.

Adjustment of water activity of media for comparative studies
The media used in this study was Malt Extract Agar (MEA).Water activity of media was modified to five water activity levels (0.98, 0.95, 0.93, 0.90 and 0.85 aw) by the addition of ionic solute, sodium chloride (NaCl) or the non-ionic solute, glycerol (Dallyn and Fox, 1980) and a control was unmodified media with freely available water (0.995aw).All the media were sterilized by autoclaving at 121°C, for 20 min.The aw levels were kept constant by keeping the same aw treatments in polyethylene bags and checked using a AQUALAB ® 3TE, USA.

Inoculation of fungi and incubation conditions
The spores of the four strains were gently dislodged from the colony surface with a sterile spatula suspended in 10 ml of sterile distilled water containing 0.05% Tween-20 in 25 ml bottles.The spore suspensions prepared could be stored frozen for 5 days or more without loss of viability.The fungal spore concentration was determined using a haemocytometer and adjusted to 10 6 spores ml - 1 .Petri plates with NaCl and glycerol-modified MEA were inoculated with 5 µl of the spore suspension and incubated at 25°C.Three replicates pre-treatment were used along with all experiments to ensure repetability.Those at the same water activity level were placed in sealed polyethylene bags (Marin et al., 1995).

Measurement of fungal growth
The diameters of fungal colony of three replicate plates were measured in two directions at right angles to each other.The growth measurements were recorded daily until the Petri plates were completely covered by the fungal grwoth (Aldred et al., 1999) and the growth rate was calculated by plotting radial mycelium growth vs. time and radial growth rates (mm day -1 ) were calculated from the slope by linear regression (Patriarca et al., 2001).

Measurement of sporulation
The strains were inoculated on MEA medium overlaid with cellophane; this aided the entire mycelial growth to be removed.The colony was suspended in 10ml of sterile water containing a wetting agent (Tween 80, 0.1%) to wet the spores (Ramos et al., 1999).Fungal spores were filtered and collected through sterile glass wool, and the filtrate was centrifuged to obtain a spore pellet.The number of spores was determined per centimetre (cm) of colony at the end of incubation period using a haemocytometer and a microscope (Ramos et al., 1999).

Effects of water activity on growth rate
Water activity is the most important factor determining growth, evolution and adhesion degree of moulds on nutrient substrates (Bouras et al., 2009).Figures 1 and 2 compares the growth rate of A. flavus, A. ochraceus, A. carbonarius and P. verrucosum at different a w levels modified by glycerol and NaCl respectively.The maximum growth rate was achieved at 0.98 a w for all species using both solutes except for A. flavus, it was at 0.995 a w in comparison to NaCl modified media.However, when water stess was applied, there was a slower growth for all species until 0.9 a w , there was no growth for A. carbonarius using NaCl solute and 0.85 a w for A. carbonarius and A. flavus using glycerol solute.A. ochraceus and P. verrucosum had a higher tolerance to lower water activity than A. carbonarius and A. flavus when modified with the NaCl solute.In all cases the a w range of 0.98-0.93resulted in a faster growth rate when compared to the control (0.995 a w ) except A. flavus using glycerol solute.The use of glycerol to modify media water availability produced a higher growth rate than with NaCl, probably because it can be utilized as a carbon and energy source and can act directly as a compatible solute (Parra et al., 2004).In contrast, high concentrations of NaCl can be toxic and this may explain the differential growth patterns observed.Belli et al. (2004) who reported optimal growth of isolates of Aspergillus section Nigri on synthetic grape juice medium between 0.95 and 0.995 a w found similar results.They also observed a similar tendency in growth rates of the isolates, which increased with a w , reaching the optimum at 0.98 a w and decreasing slightly at 0.995 a w .Previously, it was reported that the minimum value of a w required for fungal growth is in range of 0.65 -0.77.Mitchell et al. (2005) reported that the optimum value of a w for growth of A. carbonarius is in the range of 0.93 -0.9 while for A. niger a w is 0.97 (Esteban et al., 2006).The value of a w for fungi growth varies depending on temperature conditions, type of substrate and the region, in which fungi were detected.Recently, Lahouar et al. (2016) reported that maximum diameter growth rates were observed at 0.99 a w at 37°C for two toxinogenic A. flavus isolates.The minimum a w needed for mycelial growth was 0.91 at 25 and 37°C.

Effects of water activity on asexual sporulation
There were significant differences in sporulation between species on glycerol and NaCl-amended media.The  optimum conditions for production of asexual spores is often very different from that for growth.With the exception of A. carbonarius and P. verrucosum, oftenmaximum spores were produced at sub-optimal water availability conditions, regardless of solute used.A. flavus produced high concentrations of spores at 0.95 a w on modified media with glycerol and NaCl (Figure 3) , while A. ochraceus had markedly high sporulation capacities at 0.93 a w (Figure 4).There was a dramatically decrease in spores production by P. verrucosum when water stress  imposed 0.98-0.85aw with NaCl amended media and 0.95-0.85with glycerol media (Figure 5).In case of A.
carbonarius, the highest amount of spores was produced at control media (0.995 a w ) and no spore production at 0.93-0.9 a w with NaCl media (Figure 6).This may be due to osmoregulation which is an energy-requiring process and may affect sporulation process.Our results showed that there was a good relationship between growth and sporulation in case of A. carbonarius and P. verrucosum with glycerol media where the optimum a w for growth and sporulation was 0.98-0.99.While, there was no relation in case A. ochraceus and A. flavus.A study done by Gervais and Molin (2003) with P. roquefortii strains from cheese grown optimally at 0.97-0.98 a w , while maximum spore production was at 0.96 a w .Parra et al. (2004) showed that the highest amount of spores produced by a genetically engineered A. niger strain was at 0.95 a w when this was modified with glycerol at 35°C, and by a wild-type strain of A. niger at 0.97 a w and 35°C.Giorni et al. (2008) found that maximum number of spores from strains of A. flavus (from Italy) was produced at 0.96 a w .Recently, Thomas and Ogunkanmi (2014) showed that the highest amount of conidia produced by A. carbonarius was at 0.95 a w followed by 0.99 a w and 0.98 a w at all temperatures examined.The relationship between sporulation and mycotoxin production was documented by Mostafa et al. (2005) who determined that most of the mycotoxins produced after the completion of initial fungal growth followed by developmental stage, represented by sporulation, and sclerotial formation.Another study by Atoui et al. (2007) on A. carbonarius strains suggests that a significant percentage of the mycotoxin ochratoxin A, was channelled into the conidia, and this varied with environmental stress.

Conclusion
From this study the growth rate and sporulation of four mycotoxigenic fungi (A.flavus, A. ochraceus, A. carbonarius and P. verrucosum), several parameters important for maximizing the growth rate and sporulation have been identified.Water activity had the greatest effect on growth rate and sporulation.A. flavus grew significantly better than the other strains examined, while A. ochraceus produced higher amount of asexual spores than other strains.For developing control systems on prevention of food contamination by mycotoxigenic fungi and their toxins, it is necessary to study factors influencing on fungal growth and spore production.Controlling water activity will help objectively forecast the contamination level of dried product by fungi and mycotoxins.

CONFLICT OF INTERESTS
The author has not declared any conflict of interests.

REFERENCES
Aldred D, Magan N, Lane BS (1999).Influence of water activity and nutrient on growth and production of squalestatin S1 by a Phoma sp.

Figure 1 .
Figure 1.Effect of water activity modified with glycerol on growth of four toxigenic fungi.

Figure 2 .
Figure 2. Effect of water activity modified with NaCl on growth of four toxigenic fungi.

Figure 3 .
Figure 3. Water activity effects on spore production of A. flavus on MEA medium.

Figure 4 .
Figure 4. Water activity effects on spore production of A. ochraceus on MEA medium.

Figure 5 .
Figure 5.Effect of water activity on sporulation of P. verrucosum on MEA medium.

Figure 6 .
Figure 6.Effect of water activity on sporulation of A. carbonarius on MEA medium.