African Journal of
Microbiology Research

  • Abbreviation: Afr. J. Microbiol. Res.
  • Language: English
  • ISSN: 1996-0808
  • DOI: 10.5897/AJMR
  • Start Year: 2007
  • Published Articles: 5254

Full Length Research Paper

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

Ahmed Mustafa Abdel-Hadi
  • Ahmed Mustafa Abdel-Hadi
  • Botany and Microbiology Department, Faculty of Science, Al-Azhar University, Assiut Branch, Egypt.
  • Search for this author on:
  • Google Scholar


  •  Received: 24 February 2017
  •  Accepted: 29 March 2017
  •  Published: 21 April 2017

 ABSTRACT

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.

Key words: Food fungi, osmotic, abiotic parameters, conidiospores


 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 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 (aw), 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 aw 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 aw 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 aw 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 aw x temperature conditions (Cairns-Fuller et al., 2005). Generally, most moulds do not grow at a water activity (aw) 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 aw modified by glycerol and NaCl on growth rate, and a sexual spore production of four mycotoxigenic strains.


 MATERIALS AND METHODS

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 106 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).


 RESULTS AND DISCUSSION

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 aw levels modified by glycerol and NaCl respectively. The maximum growth rate was achieved at 0.98 aw for all species using both solutes except for A. flavus, it was at 0.995 aw in comparison to NaCl modified media. However, when water stess was applied, there was a slower growth for all species until 0.9 aw, there was no growth 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 the NaCl solute. In all cases the aw range of 0.98-0.93 resulted in a faster growth rate when compared to the control (0.995 aw) 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 aw found similar results. They also observed a similar tendency in growth rates of the isolates, which increased with aw, reaching the optimum at 0.98 aw and decreasing slightly at 0.995 aw. Previously, it was reported that the minimum value of aw required for fungal growth is in range of 0.65 - 0.77. Mitchell et al. (2005) reported that the optimum value of aw for growth of A. carbonarius is in the range of 0.93 - 0.9 while for A. niger aw is 0.97 (Esteban et al., 2006). The value of aw 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 aw at 37°C for two toxinogenic A. flavus isolates. The minimum aw 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, often-maximum spores were produced at sub-optimal water availability conditions, regardless of solute used. A. flavus produced high concentrations of spores at 0.95 aw on modified media with glycerol and NaCl (Figure 3), while A. ochraceus had markedly high sporulation capacities at 0.93 aw (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 0.95-0.85 with glycerol  media  (Figure 5).  In  case  of  A. carbonarius, the highest amount of spores was produced at control media (0.995 aw) and no spore production at 0.93-0.9 aw 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 aw 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 aw, while maximum spore production was at 0.96 aw. Parra et al. (2004) showed that the highest amount of spores produced by a genetically engineered A. niger strain was at 0.95 aw when this was modified with glycerol at 35°C, and by a wild-type strain of A. niger at 0.97 aw 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 aw. Recently, Thomas and Ogunkanmi (2014) showed that the highest amount of conidia produced by A. carbonarius was at 0.95 aw followed by 0.99 aw and 0.98 aw 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. J. Appl. Microbiol. 87:842-848.
Crossref
 

Astoreca A, Vaamonde G, Dalcero A, Ramos, AJ, Marin, S (2012). Modelling the effect of temperature and water activity of Aspergillus flavus isolates from corn. Int. J. Food Microbiol. 156:60-67.
Crossref
 

Atoui A, Mitchell D, Mathieu F, Magan N, Lebrihi A (2007). Partitioning of ochratoxin A in mycelium and conidia of Aspergillus carbonarius and the impact on toxin contamination of grapes and wine. Appl. Microbiol. 103:961-969.
Crossref
 

Belli N, Marin, S, Sanchis V, Ramos AJ (2004). Influence of water activity and temperature on growth of isolates of Aspergillus section Nigri obtained from grapes. Int. J. Food Microbiol. 96:19-27.
Crossref
 

Bouras N, Kim YM, Strelkov SE (2009). Influence of water activity and temperature on growth and mycotoxin production by isolates of Pyrenophora tritici-repentis from Wheat. Int. J. Food Microbiol. 131:251-255.
Crossref
 

Cairns-Fuller V, Aldred D, Magan N (2005). Water, temperature and gas composition interactions affect growth and ochratoxin A production by isolates of Penicillium verrucosum on wheat grain. J. Appl. Microbiol. 99:1215-1221.
Crossref
 

Dallyn H, Fox A (1980). Spoilage of material of reduced water activity by xerophilic fungi. In: Gould GH, Corry JEL (eds), Society of Applied Bacteriology Techincal Series 15:129-139.
 

Dorner JW, Cole RJ, Lomax LG, Gosser HS, Diener U (1983). Cyclopiazonic acid production by Aspergillus flavus and its effects on broiler chickens. Appl. Environ. Microbiol. 983:698-703.
 

Esteban A, Abarca ML, Bragulat MR, Cabanes FJ (2006). Effect of water activity on ochratoxin A production by Aspergillus niger aggregate species. Int. J. Food Microbiol. 108:188-195.
Crossref
 

Garcia D, Ramos AJ, Sanchis V, Marin S (2011). Modelling the effect of temperature and water activity in the growth boundaries of Aspergillus ochraceus and Aspergillus parasiticus. Food Microbiol. 28:406-417.
Crossref
 

Gervais P, Molin P (2003). The role of water in solid-state fermentation. Biochem. Eng. J. 13:85-101.
Crossref
 

Giorni P, Battilani P, Pietri A, Magan N (2008). Effect of aw and CO2 level of Aspergillus flavus growth and aflatoxin production in high moisture maize post-harvest. Int. J. Food Microbiol. 122: 109-113.
Crossref
 

JECFA (1997). Evaluation of certain food additives and contaminants. Forty-sixth Report of the Joint FAO/WHO Expert Committee on Food Additives 1996. : WHO Technical Report Series, 868. World Health Organization, Geneva.
 

Konietzny U, GreinerR (2003). The application of PCR in detection of mycotoxigenic fungi in food. Braz. J. Microbiol. 34:283-300.
Crossref
 

Lahouar A, Marin S, Crespo-Sempere A, Said S, Sanchis V (2016). Effects of temperature, water activity and incubation time on fungal growth and aflatoxin B1 production by toxinogenic Aspergillus flavus isolates on sorghum seeds. Rev Argent Microbiol. 48:78-85.
Crossref
 

Magan N, Aldred D (2007). Post-harvest control strategies: minimizing mycotoxins in the food chain. Int. J. Food Microbiol. 119:131-139.
Crossref
 

Marín S, Sanchis V, Magan N (1995). Water activity, temperature and pH effects on growth of Fusarium moniliforme and Fusarium proliferatum isolates from maize, Can. J. Microbiol. 41: 1063-1070.
Crossref
 

Mitchell D, Parra R, Aldred D, Magan N (2004). Water and temperature relations of growth and ochratoxin A production by Aspergillus carbonarius strains from grapes in Europe and Israel. J. Appl. Microbiol. 97:439-445.
Crossref
 

Mostafa ME, Barakat A, El Shanawany AA, (2005). Relationship between aflatoxin synthesis and Aspergillus flavus development. Afr. J. Mycol. Biotechnol. 13:35-51.
 

Pardo E, Lagunas U, Sanchis V, Ramos A, Marín S (2005). Influence of water activity and temperature on conidial germination and mycelial growth of ochratoxigenic isolates of Aspergillus ochraceus on grape juice synthetic medium. Predictive Models J. Sci. Food Agr. 85:1681-1686.
Crossref
 

Parra R, Aldred D, Archer D, Magan N (2004). Water activity, solute and temperature modify growth and spore production of wild type and genetically engineered Aspergillus niger strains. Enzyme Microb. Technol. 35:232-237.
Crossref
 

Patriarca A, Vaamonde G, Fernandez V, Comerio R (2001). Influence of water activity and temperature on the growth of Wallemia sebi: application of a predictive model. Int. J. Food Microbiol. 68:61-67.
Crossref
 

Pitt JI, Hocking AD (1997). Fungi and Food Spoilage, 2nd edn. Blackie Academic and Professional, London.
Crossref
 

Plestina R (1996). Nephrotoxicity of ochratoxin A. Food Addit. Contam. 13:49-50.
 

Ramos AJ, Sanchis V, Magan N (1999). Osmotic and matric potential effects on growth, sclerotia and partitioning of polyols and sugars in colonies and spores of Aspergillus ochraceus. Mycol Res. 103:141-147.
Crossref
 

Rao LB, Husain, A (1985). Presence of cyclopiazonic acid in kodo millet (Paspalum scrobiculatum) causing 'kodua poisoning' in man and its production by associated fungi. Mycopathologia 89:177-180.
Crossref
 

Sanchis V, Magan N (2004). Environmental conditions affecting mycotoxins. In: Magan N, Olsen M (eds), Mycotoxins in food 174-189.
 

Sinha RN (1995). The stored grain ecosystemse. In Stored Grain Ecosystems. Edited by Jayas DS, White NDG, Muir WE. Marcell Dekker. 1-32.
 

Thomas, BT, Ogunkanmi LA (2014). Influence of Water Activity and Temperature on the Growth and Accumulation of Ochratoxin A Produced by Aspergillus carbonarius Isolated from Garri. Int. J. Microbiol. Res. 3:152-159.

 




Copyright © 2025 Author(s) retain the copyright of this article.

This article is published under the terms of the Creative Commons Attribution License 4.0

Back to Vol. 11 No. 15
Back to articles
SUBSCRIBE TO RSS
SUBMIT MANUSCRIPT
CONFERENCES
          */?>