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Impact of water activity of diverse media on spore germination of Aspergillus and Penicillium species
International Journal of Food Microbiology 142 (2010) 273–276
Contents lists available at ScienceDirect
International Journal of Food Microbiology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / i j f o o d m i c r o
Short communication
Impact of water activity of diverse media on spore germination of Aspergillus and Penicillium species Sidjè Paule-Marina Nanguy a, Jean-Marie Perrier-Cornet a, Maurice Bensoussan b, Philippe Dantigny a,⁎ a b
Laboratoire de Génie des Procédés Microbiologiques et Alimentaires, Agro-Sup Dijon, 1 Esplanade Erasme, 21000 Dijon, France GTR Mycologie, Sécurité Microbiologique Alimentaire et Environnement, Université de Bourgogne, Agro-Sup Dijon, France
a r t i c l e
i n f o
Article history: Received 7 May 2010 Received in revised form 27 June 2010 Accepted 30 June 2010 Keywords: Water activity Germination Aspergillus carbonarius Aspergillus flavus Penicillium chrysogenum Penicillium expansum
a b s t r a c t The effects of water activity (aw) of diverse media i/ culture medium for sporogenesis, aw sp ii/ liquid spore suspension medium, aw su and iii/ medium for germination, aw ge, on the germination time tG of Aspergillus carbonarius, Aspergillus flavus, Penicillium chrysogenum and Penicillium expansum were assessed according to a screening matrix at 0.95 and 0.99 aw. It was shown that i/ reduced tGs were obtained at 0.95 aw sp except for P. expansum ii/ a significant effect of aw su on tG was demonstrated for A. carbonarius, P. chrysogenum and P. expansum iii/ the most important factor for controlling the germination time was the medium for germination except for A. carbonarius (aw su). In accordance with the fact that fungal spores can swell as soon as they are suspended in an aqueous solution it is recommended to re-suspend fungal spores in a solution at the same water activity as that of subsequent germination studies. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Fungal spore germination marks the resumption of vegetative development and the formation of a new individual or colony (Isaac, 1998). Germination of a mould spore is a physiological reaction of a resting cell to changes in environmental conditions. This process can conveniently be divided into two stages: a period of an increase in the spore size (swelling phase), followed by the emergence of the germ tube (outgrowth phase) (Garraway and Evans, 1984). Hydration is a predominant part of germination, and the water activity of the medium is an important factor (Beuchat, 1983, 1987; Pitt, 1989). The standardized protocol for producing spores prior to assessing the effect of the water activity during germination can be summarized as follows. Fresh spores are obtained after mycelium has been grown on various semi-synthetic media (0.99 aw) such as Potato Dextrose Agar (PDA) or Malt Extract Agar (MEA), (Dantigny et al., 2006). Then, spore suspensions are obtained by flooding the mycelium with a saline (NaCl, 9 g/L) aqueous solution that contained Tween 80, a wetting agent. However, post-harvest re-suspension of fungal spores in saline solution (0.99 aw) might interfere with subsequent studies carried out at different water activities. In order to avoid these possible interferences, a stock spore suspension of Penicillium verrucosum was diluted in a sterile water/glycerol solution to the required water availability of subsequent experiments (Pardo et al., 2006). More
⁎ Corresponding author. Tel.: + 33 3 80 77 40 71; fax: + 33 3 80 39 66 40. E-mail address: [email protected] (P. Dantigny). 0168-1605/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.ijfoodmicro.2010.06.031
recently, the conidia of Penicillium chrysogenum were harvested by flooding the surface of the plates with a solution at the same water activity as subsequent germination studies (Judet et al., 2008). Similarly, the conidia of Aspergillus flavus were harvested using a sterile glycerol-water solution with the aw adjusted to match that of the growth medium (Alam et al., 2010). The impact of water-stress and hypo-osmotic and iso-osmotic washing treatments on germinability of Metarhizium anisoplae was studied previously. It has been mentioned that, when these fungi were grown under low environmental water potentials (water-stress conditions), they were able to lower their intracellular water potential by an increased synthesis and/or accumulation of polyols such as glycerol and erythritol (Hallsworth et al., 2003). Any subsequent exposure of such modified structures to an external environment with a water potential different from the intracellular water potential would subject these structures to an osmotic shock (Ypsilos and Magan, 2004). This implies that the water activity had not a trivial effect. Sporulation conditions and re-hydration had a great impact on the physiological state of fungal spores (Dantigny and Nanguy, 2009). Our understanding of the way in which the stress parameter water activity impacts on distinct phases of the life cycle of Ascomycete fungi is, as yet, incomplete. When water activity for sporulation and germination are different, what should be the water activity of spore suspension to avoid bias in the determination of the germination time? The aim of this study was to compare the effects of water activity (aw) of diverse media i/ culture medium for sporogenesis ii/ liquid spore suspension medium and iii/ medium for germination, on the germination time of Aspergillus carbonarius, A. flavus, P. chrysogenum and Penicillium expansum.
274
S.P.-M. Nanguy et al. / International Journal of Food Microbiology 142 (2010) 273–276
2. Materials and methods 2.1. Moulds and media A. carbonarius was provided by the Institute for Vine and Wine, (IUVV, Dijon, France), A. flavus MUCL 1890 was obtained from the Mycothèque Universitaire de Louvain, Belgique, P. chrysogenum 738 was isolated from baking products and P. expansum 25.3 was isolated from a vineyard in Burgundy. All moulds were maintained on potato dextrose agar (PDA) medium (bioMérieux, Marcy l'Etoile, France) at room temperature (18 to 25 °C). The strains are stored at the laboratory and are available under request. The media for spore production and spore germination was PDA. The initial pH for all experiments was 5.7 ± 0.1. Water activity (aw) in these media was adjusted by substituting part of the water with an equal weight of glycerol (Gervais et al., 1988). Glycerol can act as a stressor at molar concentrations (Williams and Hallsworth, 2009). The concentrations used for spore suspensions (2.2 M at 0.95 aw) were clearly above this level but did not reach chaotropic concentrations greater than equal to 6 M.
measured by means of a Leica DMLB (x200) (Leica, Rueil-Malmaison, France) connected to an IXC 800 (I2S, Pessac, France) camera. Pictures were analyzed using Matrox Inspector 2.2 (Matrox Electronics Systems Ltd, Dorval, Canada). Spores were considered germinated when the length of the germ tubes was equal to the greatest dimension of the swollen spore (Dantigny et al., 2006). A logistic model (Withing and Call, 1993; Dantigny et al., 2002, 2006; Judet et al., 2008) was used for describing the percentage of germinated spores as a function of time: kðτ−tÞ P = Pmax = 1 + e
ð1Þ
Where P (%) was the percentage of germinated spores, Pmax (%) the maximum percentage, k (h−1) was the slope term for the rate of increasing germinated spores, τ (h) was the time of the inflection point where P equals half to the Pmax and t was the time (h). Germination time was defined as the time at which 50% of viable spores germinated. By using this definition, tG was equal to τ. 2.5. Analysis and interpretation of the results
2.2. Spore production The PDA medium was adjusted to 0.95 aw sp and 0.99 aw sp. The plates were single-point inoculated and incubated at 25 °C for 7 d. Spores were collected by flooding the surface of the plates with 4.5 ml of sterile saline solution (NaCl, 9 g/l of water) containing Tween 80 (0.05% vol/vol; Prolabo, Paris, France) and glycerol to the set values 0.95 aw su and 0.99 aw su. After counting the spores on a Malassez cell, the spore suspensions were standardized to 1 × 106 spores ml−1.
All parameters were estimated by nonlinear regression software based upon the Levenberg–Marquardt Algorithm (Slidewrite 5.0, Advanced Graphics Software, Inc, Carlsbad, CA, USA). Multiple regression analysis based on the latest square method was performed using Nemrod software (LPRAI, Marseille, France). A linear polynomial equation was used to describe the main effects of the factors, aw sp, aw su and aw ge on the germination time. The respective coded values X1, X2 and X3 were equal to −1 (0.95 aw) and to +1 (0.99 aw).
2.3. Spore germination
tG = b0 + b1 ⋅X1 + b2 ⋅X2 + b3 ⋅X3
The device used in this study was made from a Petri dish and was described previously (Sautour et al., 2001). The PDA medium was adjusted to 0.95 aw ge and 0.99 aw ge and was poured on the internal side of the lid of the Petri dish. In order to equilibrate the relative humidity inside each device after inoculation, an appropriate water/ glycerol solution (15 ml) was poured into the Petri dish. The water activity of this solution was identical to that of the culture medium tacked to the lid. The devices sealed with Parafilm® constituted the closed incubation chambers. After solidification, the medium was inoculated with 10 μl of the standardized suspensions.
The significance of the coefficients was evaluated by multiple regression analysis based upon the F-test with unequal variance.
2.4. Assessment of germination Without opening the devices, at least 100 spores (20–25 per microscopic field) were examined through the Petri dish lid every hour. Experiments were carried out in duplicate. Germination temperature was 20 ± 1 °C °C. The length of the germ tubes was
ð2Þ
3. Results The germination time, tG, was reproducible. The differences between the duplicates were less than or equal to 0.2 h. The germination time was also accurately determined by the logistic model. Standard errors were in the range 0.05–0.25 h (data not shown). With the exception of A. carbonarius, the greatest tG values were obtained when germination was assessed at 0.95 aw ge whatever the other conditions, Table 1. Conversely, the shortest tG values were shown when germination occurred at 0.99 aw ge (Figs. 1–4). This is consistent with negative b3 coefficients. For X3 = + 1 (i.e., 0.99 aw ge), the term b3 · X3 was negative, thus leading to a decrease of the experimental response tG. It was demonstrated that the water activity for germination was the most important factor by comparing the
Table 1 Experimental protocol for screening the influence of the water activity of diverse media (culture medium for sporogenesis, liquid spore suspension medium and medium for germination) on the germination time of Aspergillus carbonarius, Aspergillus flavus, Penicillium chrysogenum and Penicillium expansum. The two germination times are duplicate data (number of spores examined). Water activity (aw) of diverse media
Germination time (h)
Culture medium for sporogenesis
Liquid spore suspension
Medium for germination
Aspergillus carbonarius
Aspergillus flavus
Penicillium chrysogenum
Penicillium expansum
0.99
0.99
0.95
0.95
0.99
0.99
0.99
0.95
0.99
0.95
0.95
0.95
12.6 12.4 10.3 10.3 12.6 12.6 13.4 13.4
32.3 32.4 13.4 13.4 20.1 20.3 25.4 25.4
16.7 16.8 11.1 11.3 12.3 12.3 17.4 17.3
16.9 16.8 13.3 13.3 15.0 15.0 20.4 20.4
(100) (111) (113) (120) (100) (104) (100) (102)
(108) (110) (100) (112) (108) (109) (114) (106)
(120) (115) (108) (107) (100) (111) (100) (108)
(109) (100) (100) (108) (100) (105) (100) (100)
S.P.-M. Nanguy et al. / International Journal of Food Microbiology 142 (2010) 273–276
Fig. 1. Germination curves obtained for Aspergillus carbonarius at (●) 0.99 aw sp; 0.99 aw su; 0.95 aw ge; (▲) 0.95 aw sp; 0.99 aw su; 0.99 aw ge; (Δ) 0.99 aw sp; 0.95 aw su; 0.99 aw ge; (○) 0.95 aw sp; 0.95 aw su; 0.95 aw ge. Logistic model (—).
275
Fig. 3. Germination curves obtained for Penicillium chrysogenum at (●) 0.99 aw sp; 0.99 aw su; 0.95 aw ge; (▲) 0.95 aw sp; 0.99 aw su; 0.99 aw ge; (Δ) 0.99 aw sp; 0.95 aw su; 0.99 aw ge; (○) 0.95 aw sp; 0.95 aw su; 0.95 aw ge. Logistic model (—).
absolute values of the coefficients. It was shown that |b3| was greater than |b1| and |b2| for all the species except for A. carbonarius. For this species, |b2| was the greater coefficient (Table 2), the effect of the water activity of the liquid suspension was greater than the effect of the water activity of the germination medium. Therefore the germination time at the optimum aw su 0.99/0.99/0.95 (12.6 and 12.4 h) was slightly less than that at the optimum aw ge 0.99/0.95/0.99 (12.6 and 12.6 h). The response means represented the estimated germination time for all coded factors equaled to zero (i.e., 0.975 aw). The response mean was the greatest for A. flavus and the least for A. carbonarius. This was consistent with germination times which were the greatest for A.
flavus and the least for A. carbonarius in all the experimental conditions except at 0.99 aw sp, 0.95 aw su, 0.99 aw ge, Table 1. The coefficients b1 were significant for all the species and positive for all the species except P. expansum. For positive b1 coefficients, tG was decreased when sporogenesis occurred at 0.95 aw sp. In fact, for a positive value of b1, the term b1 · X1 was negative for X1 = −1 (i.e., 0.95 aw sp). In contrast to A. flavus, the water activity of the spore suspension, aw su, was significant for A. carbonarius, P. chrysogenum and P. expansum, Table 2. It was noticed for these three species that |b2| was greater than |b1|, thus highlighting that the water activity of the spore suspension had a greater effect on tG, than the water activity during sporogenesis. More importantly, the signs of the coefficients b2 and b3 were identical thus demonstrating that the water activity of the spore
Fig. 2. Germination curves obtained for Aspergillus flavus at (●) 0.99 aw sp; 0.99 aw su; 0.95 aw ge; (▲) 0.95 aw sp; 0.99 aw su; 0.99 aw ge; (Δ) 0.99 aw sp; 0.95 aw su; 0.99 aw ge; (○) 0.95 aw sp; 0.95 aw su; 0.95 aw ge. Logistic model (—).
Fig. 4. Germination curves obtained for Penicillium expansum at (●) 0.99 aw sp; 0.99 aw su; 0.95 aw ge; (▲) 0.95 aw sp; 0.99 aw su; 0.99 aw ge; (Δ) 0.99 aw sp; 0.95 aw su; 0.99 aw ge; (○) 0.95 aw sp; 0.95 aw su; 0.95 aw ge. Logistic model (—).
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Table 2 List of model coefficients. Factors
response means aw sporogenesis aw suspension aw germination
Coefficients
b0 b1 b2 b3
Aspergillus carbonarius
Aspergillus flavus
Penicillium chrysogenum
Penicillium expansum
Value
p-value
Value
p-value
Value
p-value
Value
p-value
12.2 0.35 −0.80 −0.75
b 0.01 0.0574 0.0154 0.0166
22.8 3.41 0.01 −6.01
b0.01 b0.01 25.1 b0.01
14.4 0.13 −0.42 −2.65
b0.01 1.63 0.0584 b0.01
16.4 −0.46 −1.31 −2.24
b 0.01 0.0131 b 0.01 b 0.01
suspension should be identical to that during germination. The coefficients b2 and b3 were negative, but a similar demonstration could be done for both the coefficients were positive. A negative b3 coefficient implies an increase of tG at 0.95 aw ge. An additional increase of tG is obtained if the water activity of the suspension is also 0.95. Otherwise there is an antagonistic effect between the water activity of the suspension, aw su, and the water activity of germination, aw ge. For example, assuming 0.95 aw su and 0.99 aw ge, the coded factors are X2 = −1 and X3 = + 1. In such a condition, the terms b2 · X2 and b3 · X3 are positive and negative respectively. Therefore, there is an antagonistic effect because the spore suspension at 0.95 aw tends in an increase of tG whereas the germination at 0.99 aw tends in a decrease of tG.
characterized by small germination time such as A. carbonarius are more susceptible to the water activity of the suspension than those characterized by longer germination time such as A. flavus. It was highlighted for A. carbonarius that the effect of the water activity of the liquid spore suspension on the germination time was more important than that of the water activity of the germination medium. The data obtained for Aspergillus and Penicillium species suggest that the water activity of media used to prepare spore suspensions should be identical to that of subsequent germination studies, because the germination process is initiated as soon as conidia are in contact with a solution (Dantigny et al., 2006).
References 4. Discussion A screening matrix was used to determine the main effects of three factors on the germination time of A. carbonarius, A. flavus, P. chrysogenum and P. expansum. Accordingly a simple four parameter polynomial model was used. However, the combined effects were not assessed. Combined effects occurred when the effect of one factor depended upon the level of another one. It was shown previously that the effect of a reduced water activity during sporogenesis on the germination of P. chrysogenum was greater at 0.95 aw ge than at 0.99 aw ge (Judet et al., 2008). Therefore, interaction between the water activity during sporogenesis and during germination probably occurred in the present study. Accordingly, the proposed model cannot be used for predicting the germination time in other experimental conditions than those described here. In order to quantify the combined effects, a full factorial matrix should be used. Four other experiments should be carried out in addition to the four experiments already described in this study. As compared to conidia produced at 0.99 aw sp, the germination time of all the species except P. expansum was shortened for conidia produced at 0.95 aw sp. It was suggested that the enhancement of the germination of conidia obtained under osmotic stress may be due to a greater gradient of water across the cell membrane (Judet et al., 2008). Dormant spores are characterized by a low hydration and prior to germination most species require the presence of liquid water or high relative humidity. During activation, large amounts of free water will enter into the spore, rapidly at first, leading to spore swelling (Isaac, 1998). The enhancement of conidial germination due to synthesis and/or accumulation of glycerol and erythritol were shown for several species under NaCl-induced osmotic stress (Hallsworth and Magan, 1995) and for A. nidulans under ethanol and NaCl-induced osmotic stress (Hallsworth et al., 2003). This study suggested that the water activity of suspension had a greater effect on germination time than did the kind of mould. Moulds
Alam, S., Shah, H.U., Magan, N., 2010. Effect of calcium propionate and water activity on growth and aflatoxins production by Aspergillus flavus. J. Food Sci. 75, M61–M64. Beuchat, L.R., 1983. Influence of water activity on growth, metabolic activities and survival of yeast and molds. J. Food Prot. 46, 135–141. Beuchat, L.R., 1987. Influence of water activity on sporulation, germination, outgrowth and toxin production. In: Rockland, L.B., Beuchat, L.R. (Eds.), Water Activity: Theory and Applications to Food. Marcel Dekker Inc, New York, pp. 137–151. Dantigny, P., Nanguy, S.P.M., 2009. Significance of the physiological state of fungal spores. Int. J. Food Microbiol. 134, 16–20. Dantigny, P., Soares Mansur, C., Sautour, M., Tchobanov, I., Bensoussan, M., 2002. Relationship between spore germination kinetics and lag time during growth of Mucor racemosus. Lett. Appl. Microbiol. 35, 395–398. Dantigny, P., Bensoussan, M., Vasseur, V., Lebrihi, A., Buchet, C., Ismaili-Alaoui, M., Devlieghere, F., Roussos, S., 2006. Standardisation of methods for assessing mould germination: a workshop report. Int. J. Food Microbiol. 108, 286–291. Garraway, M.O., Evans, R.C., 1984. Fungal Nutrition and Physiology. Wiley, New York. Gervais, P., Fasquel, J.-P., Molin, P., 1988. Water relations of spore germination. Appl. Microbiol. Biotechnol. 29, 586–592. Hallsworth, J.E., Magan, N., 1995. Manipulation of intracellular glycerol and erythritol enhances germination of conidia at low water availability. Microbiology 141, 1109–1115. Hallsworth, J.E., Prior, B.A., Nomura, Y., Iwahara, M., Timmis, K.N., 2003. Compatible solutes protect against chaotrope (ethanol)-induced, nonosmotic water stress. Appl. Environ. Microbiol. 69, 7032–7034. Isaac, S., 1998. What factors influence the germination and outgrowth of fungal spores? Mycologist 12 (Part 2, 91). Judet, D., Bensoussan, M., Perrier-Cornet, J-M., Dantigny, P., 2008. Distributions of the growth rate of the germ tubes and germination time of Penicillium chrysogenum conidia depend on water activity. Food Microbiol. 25, 902–907. Pardo, E., Malet, M., Marín, S., Sanchis, V., Ramos, A.J., 2006. Effects of water activity and temperature on germination and growth profiles of ochratogenic Penicillium verrucosum isolates on barley meal extract agar. Int. J. Food Microbiol. 106, 25–31. Pitt, J.I., 1989. Water activity, the neglected parameter. Aust. Microbiol. 10, 93–97. Sautour, M., Rouget, A., Dantigny, P., Divies, C., Bensoussan, M., 2001. Prediction of conidial germination of Penicillium chrysogenum as influenced by temperature, water activity and pH. Lett. Appl. Microbiol. 32, 131–134. Williams, J.P., Hallsworth, J.E., 2009. Limits of life in hostile environments: no barriers to biosphere function? Environ. Microbiol. 11, 3292–3308. Withing, R.C., Call, J.E., 1993. Time of growth model for proteolytic Clostridium botulinum. Food Microbiol. 10, 295–301. Ypsilos, I.K., Magan, N., 2004. Impact of water-stress and washing treatments on production, synthesis and retention of endogenous sugar alcohols and germinability of Metarhizium anisopliae blastospores. Mycol. Res. 108, 1337–1345.
Contents lists available at ScienceDirect
International Journal of Food Microbiology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / i j f o o d m i c r o
Short communication
Impact of water activity of diverse media on spore germination of Aspergillus and Penicillium species Sidjè Paule-Marina Nanguy a, Jean-Marie Perrier-Cornet a, Maurice Bensoussan b, Philippe Dantigny a,⁎ a b
Laboratoire de Génie des Procédés Microbiologiques et Alimentaires, Agro-Sup Dijon, 1 Esplanade Erasme, 21000 Dijon, France GTR Mycologie, Sécurité Microbiologique Alimentaire et Environnement, Université de Bourgogne, Agro-Sup Dijon, France
a r t i c l e
i n f o
Article history: Received 7 May 2010 Received in revised form 27 June 2010 Accepted 30 June 2010 Keywords: Water activity Germination Aspergillus carbonarius Aspergillus flavus Penicillium chrysogenum Penicillium expansum
a b s t r a c t The effects of water activity (aw) of diverse media i/ culture medium for sporogenesis, aw sp ii/ liquid spore suspension medium, aw su and iii/ medium for germination, aw ge, on the germination time tG of Aspergillus carbonarius, Aspergillus flavus, Penicillium chrysogenum and Penicillium expansum were assessed according to a screening matrix at 0.95 and 0.99 aw. It was shown that i/ reduced tGs were obtained at 0.95 aw sp except for P. expansum ii/ a significant effect of aw su on tG was demonstrated for A. carbonarius, P. chrysogenum and P. expansum iii/ the most important factor for controlling the germination time was the medium for germination except for A. carbonarius (aw su). In accordance with the fact that fungal spores can swell as soon as they are suspended in an aqueous solution it is recommended to re-suspend fungal spores in a solution at the same water activity as that of subsequent germination studies. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Fungal spore germination marks the resumption of vegetative development and the formation of a new individual or colony (Isaac, 1998). Germination of a mould spore is a physiological reaction of a resting cell to changes in environmental conditions. This process can conveniently be divided into two stages: a period of an increase in the spore size (swelling phase), followed by the emergence of the germ tube (outgrowth phase) (Garraway and Evans, 1984). Hydration is a predominant part of germination, and the water activity of the medium is an important factor (Beuchat, 1983, 1987; Pitt, 1989). The standardized protocol for producing spores prior to assessing the effect of the water activity during germination can be summarized as follows. Fresh spores are obtained after mycelium has been grown on various semi-synthetic media (0.99 aw) such as Potato Dextrose Agar (PDA) or Malt Extract Agar (MEA), (Dantigny et al., 2006). Then, spore suspensions are obtained by flooding the mycelium with a saline (NaCl, 9 g/L) aqueous solution that contained Tween 80, a wetting agent. However, post-harvest re-suspension of fungal spores in saline solution (0.99 aw) might interfere with subsequent studies carried out at different water activities. In order to avoid these possible interferences, a stock spore suspension of Penicillium verrucosum was diluted in a sterile water/glycerol solution to the required water availability of subsequent experiments (Pardo et al., 2006). More
⁎ Corresponding author. Tel.: + 33 3 80 77 40 71; fax: + 33 3 80 39 66 40. E-mail address: [email protected] (P. Dantigny). 0168-1605/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.ijfoodmicro.2010.06.031
recently, the conidia of Penicillium chrysogenum were harvested by flooding the surface of the plates with a solution at the same water activity as subsequent germination studies (Judet et al., 2008). Similarly, the conidia of Aspergillus flavus were harvested using a sterile glycerol-water solution with the aw adjusted to match that of the growth medium (Alam et al., 2010). The impact of water-stress and hypo-osmotic and iso-osmotic washing treatments on germinability of Metarhizium anisoplae was studied previously. It has been mentioned that, when these fungi were grown under low environmental water potentials (water-stress conditions), they were able to lower their intracellular water potential by an increased synthesis and/or accumulation of polyols such as glycerol and erythritol (Hallsworth et al., 2003). Any subsequent exposure of such modified structures to an external environment with a water potential different from the intracellular water potential would subject these structures to an osmotic shock (Ypsilos and Magan, 2004). This implies that the water activity had not a trivial effect. Sporulation conditions and re-hydration had a great impact on the physiological state of fungal spores (Dantigny and Nanguy, 2009). Our understanding of the way in which the stress parameter water activity impacts on distinct phases of the life cycle of Ascomycete fungi is, as yet, incomplete. When water activity for sporulation and germination are different, what should be the water activity of spore suspension to avoid bias in the determination of the germination time? The aim of this study was to compare the effects of water activity (aw) of diverse media i/ culture medium for sporogenesis ii/ liquid spore suspension medium and iii/ medium for germination, on the germination time of Aspergillus carbonarius, A. flavus, P. chrysogenum and Penicillium expansum.
274
S.P.-M. Nanguy et al. / International Journal of Food Microbiology 142 (2010) 273–276
2. Materials and methods 2.1. Moulds and media A. carbonarius was provided by the Institute for Vine and Wine, (IUVV, Dijon, France), A. flavus MUCL 1890 was obtained from the Mycothèque Universitaire de Louvain, Belgique, P. chrysogenum 738 was isolated from baking products and P. expansum 25.3 was isolated from a vineyard in Burgundy. All moulds were maintained on potato dextrose agar (PDA) medium (bioMérieux, Marcy l'Etoile, France) at room temperature (18 to 25 °C). The strains are stored at the laboratory and are available under request. The media for spore production and spore germination was PDA. The initial pH for all experiments was 5.7 ± 0.1. Water activity (aw) in these media was adjusted by substituting part of the water with an equal weight of glycerol (Gervais et al., 1988). Glycerol can act as a stressor at molar concentrations (Williams and Hallsworth, 2009). The concentrations used for spore suspensions (2.2 M at 0.95 aw) were clearly above this level but did not reach chaotropic concentrations greater than equal to 6 M.
measured by means of a Leica DMLB (x200) (Leica, Rueil-Malmaison, France) connected to an IXC 800 (I2S, Pessac, France) camera. Pictures were analyzed using Matrox Inspector 2.2 (Matrox Electronics Systems Ltd, Dorval, Canada). Spores were considered germinated when the length of the germ tubes was equal to the greatest dimension of the swollen spore (Dantigny et al., 2006). A logistic model (Withing and Call, 1993; Dantigny et al., 2002, 2006; Judet et al., 2008) was used for describing the percentage of germinated spores as a function of time: kðτ−tÞ P = Pmax = 1 + e
ð1Þ
Where P (%) was the percentage of germinated spores, Pmax (%) the maximum percentage, k (h−1) was the slope term for the rate of increasing germinated spores, τ (h) was the time of the inflection point where P equals half to the Pmax and t was the time (h). Germination time was defined as the time at which 50% of viable spores germinated. By using this definition, tG was equal to τ. 2.5. Analysis and interpretation of the results
2.2. Spore production The PDA medium was adjusted to 0.95 aw sp and 0.99 aw sp. The plates were single-point inoculated and incubated at 25 °C for 7 d. Spores were collected by flooding the surface of the plates with 4.5 ml of sterile saline solution (NaCl, 9 g/l of water) containing Tween 80 (0.05% vol/vol; Prolabo, Paris, France) and glycerol to the set values 0.95 aw su and 0.99 aw su. After counting the spores on a Malassez cell, the spore suspensions were standardized to 1 × 106 spores ml−1.
All parameters were estimated by nonlinear regression software based upon the Levenberg–Marquardt Algorithm (Slidewrite 5.0, Advanced Graphics Software, Inc, Carlsbad, CA, USA). Multiple regression analysis based on the latest square method was performed using Nemrod software (LPRAI, Marseille, France). A linear polynomial equation was used to describe the main effects of the factors, aw sp, aw su and aw ge on the germination time. The respective coded values X1, X2 and X3 were equal to −1 (0.95 aw) and to +1 (0.99 aw).
2.3. Spore germination
tG = b0 + b1 ⋅X1 + b2 ⋅X2 + b3 ⋅X3
The device used in this study was made from a Petri dish and was described previously (Sautour et al., 2001). The PDA medium was adjusted to 0.95 aw ge and 0.99 aw ge and was poured on the internal side of the lid of the Petri dish. In order to equilibrate the relative humidity inside each device after inoculation, an appropriate water/ glycerol solution (15 ml) was poured into the Petri dish. The water activity of this solution was identical to that of the culture medium tacked to the lid. The devices sealed with Parafilm® constituted the closed incubation chambers. After solidification, the medium was inoculated with 10 μl of the standardized suspensions.
The significance of the coefficients was evaluated by multiple regression analysis based upon the F-test with unequal variance.
2.4. Assessment of germination Without opening the devices, at least 100 spores (20–25 per microscopic field) were examined through the Petri dish lid every hour. Experiments were carried out in duplicate. Germination temperature was 20 ± 1 °C °C. The length of the germ tubes was
ð2Þ
3. Results The germination time, tG, was reproducible. The differences between the duplicates were less than or equal to 0.2 h. The germination time was also accurately determined by the logistic model. Standard errors were in the range 0.05–0.25 h (data not shown). With the exception of A. carbonarius, the greatest tG values were obtained when germination was assessed at 0.95 aw ge whatever the other conditions, Table 1. Conversely, the shortest tG values were shown when germination occurred at 0.99 aw ge (Figs. 1–4). This is consistent with negative b3 coefficients. For X3 = + 1 (i.e., 0.99 aw ge), the term b3 · X3 was negative, thus leading to a decrease of the experimental response tG. It was demonstrated that the water activity for germination was the most important factor by comparing the
Table 1 Experimental protocol for screening the influence of the water activity of diverse media (culture medium for sporogenesis, liquid spore suspension medium and medium for germination) on the germination time of Aspergillus carbonarius, Aspergillus flavus, Penicillium chrysogenum and Penicillium expansum. The two germination times are duplicate data (number of spores examined). Water activity (aw) of diverse media
Germination time (h)
Culture medium for sporogenesis
Liquid spore suspension
Medium for germination
Aspergillus carbonarius
Aspergillus flavus
Penicillium chrysogenum
Penicillium expansum
0.99
0.99
0.95
0.95
0.99
0.99
0.99
0.95
0.99
0.95
0.95
0.95
12.6 12.4 10.3 10.3 12.6 12.6 13.4 13.4
32.3 32.4 13.4 13.4 20.1 20.3 25.4 25.4
16.7 16.8 11.1 11.3 12.3 12.3 17.4 17.3
16.9 16.8 13.3 13.3 15.0 15.0 20.4 20.4
(100) (111) (113) (120) (100) (104) (100) (102)
(108) (110) (100) (112) (108) (109) (114) (106)
(120) (115) (108) (107) (100) (111) (100) (108)
(109) (100) (100) (108) (100) (105) (100) (100)
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Fig. 1. Germination curves obtained for Aspergillus carbonarius at (●) 0.99 aw sp; 0.99 aw su; 0.95 aw ge; (▲) 0.95 aw sp; 0.99 aw su; 0.99 aw ge; (Δ) 0.99 aw sp; 0.95 aw su; 0.99 aw ge; (○) 0.95 aw sp; 0.95 aw su; 0.95 aw ge. Logistic model (—).
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Fig. 3. Germination curves obtained for Penicillium chrysogenum at (●) 0.99 aw sp; 0.99 aw su; 0.95 aw ge; (▲) 0.95 aw sp; 0.99 aw su; 0.99 aw ge; (Δ) 0.99 aw sp; 0.95 aw su; 0.99 aw ge; (○) 0.95 aw sp; 0.95 aw su; 0.95 aw ge. Logistic model (—).
absolute values of the coefficients. It was shown that |b3| was greater than |b1| and |b2| for all the species except for A. carbonarius. For this species, |b2| was the greater coefficient (Table 2), the effect of the water activity of the liquid suspension was greater than the effect of the water activity of the germination medium. Therefore the germination time at the optimum aw su 0.99/0.99/0.95 (12.6 and 12.4 h) was slightly less than that at the optimum aw ge 0.99/0.95/0.99 (12.6 and 12.6 h). The response means represented the estimated germination time for all coded factors equaled to zero (i.e., 0.975 aw). The response mean was the greatest for A. flavus and the least for A. carbonarius. This was consistent with germination times which were the greatest for A.
flavus and the least for A. carbonarius in all the experimental conditions except at 0.99 aw sp, 0.95 aw su, 0.99 aw ge, Table 1. The coefficients b1 were significant for all the species and positive for all the species except P. expansum. For positive b1 coefficients, tG was decreased when sporogenesis occurred at 0.95 aw sp. In fact, for a positive value of b1, the term b1 · X1 was negative for X1 = −1 (i.e., 0.95 aw sp). In contrast to A. flavus, the water activity of the spore suspension, aw su, was significant for A. carbonarius, P. chrysogenum and P. expansum, Table 2. It was noticed for these three species that |b2| was greater than |b1|, thus highlighting that the water activity of the spore suspension had a greater effect on tG, than the water activity during sporogenesis. More importantly, the signs of the coefficients b2 and b3 were identical thus demonstrating that the water activity of the spore
Fig. 2. Germination curves obtained for Aspergillus flavus at (●) 0.99 aw sp; 0.99 aw su; 0.95 aw ge; (▲) 0.95 aw sp; 0.99 aw su; 0.99 aw ge; (Δ) 0.99 aw sp; 0.95 aw su; 0.99 aw ge; (○) 0.95 aw sp; 0.95 aw su; 0.95 aw ge. Logistic model (—).
Fig. 4. Germination curves obtained for Penicillium expansum at (●) 0.99 aw sp; 0.99 aw su; 0.95 aw ge; (▲) 0.95 aw sp; 0.99 aw su; 0.99 aw ge; (Δ) 0.99 aw sp; 0.95 aw su; 0.99 aw ge; (○) 0.95 aw sp; 0.95 aw su; 0.95 aw ge. Logistic model (—).
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Table 2 List of model coefficients. Factors
response means aw sporogenesis aw suspension aw germination
Coefficients
b0 b1 b2 b3
Aspergillus carbonarius
Aspergillus flavus
Penicillium chrysogenum
Penicillium expansum
Value
p-value
Value
p-value
Value
p-value
Value
p-value
12.2 0.35 −0.80 −0.75
b 0.01 0.0574 0.0154 0.0166
22.8 3.41 0.01 −6.01
b0.01 b0.01 25.1 b0.01
14.4 0.13 −0.42 −2.65
b0.01 1.63 0.0584 b0.01
16.4 −0.46 −1.31 −2.24
b 0.01 0.0131 b 0.01 b 0.01
suspension should be identical to that during germination. The coefficients b2 and b3 were negative, but a similar demonstration could be done for both the coefficients were positive. A negative b3 coefficient implies an increase of tG at 0.95 aw ge. An additional increase of tG is obtained if the water activity of the suspension is also 0.95. Otherwise there is an antagonistic effect between the water activity of the suspension, aw su, and the water activity of germination, aw ge. For example, assuming 0.95 aw su and 0.99 aw ge, the coded factors are X2 = −1 and X3 = + 1. In such a condition, the terms b2 · X2 and b3 · X3 are positive and negative respectively. Therefore, there is an antagonistic effect because the spore suspension at 0.95 aw tends in an increase of tG whereas the germination at 0.99 aw tends in a decrease of tG.
characterized by small germination time such as A. carbonarius are more susceptible to the water activity of the suspension than those characterized by longer germination time such as A. flavus. It was highlighted for A. carbonarius that the effect of the water activity of the liquid spore suspension on the germination time was more important than that of the water activity of the germination medium. The data obtained for Aspergillus and Penicillium species suggest that the water activity of media used to prepare spore suspensions should be identical to that of subsequent germination studies, because the germination process is initiated as soon as conidia are in contact with a solution (Dantigny et al., 2006).
References 4. Discussion A screening matrix was used to determine the main effects of three factors on the germination time of A. carbonarius, A. flavus, P. chrysogenum and P. expansum. Accordingly a simple four parameter polynomial model was used. However, the combined effects were not assessed. Combined effects occurred when the effect of one factor depended upon the level of another one. It was shown previously that the effect of a reduced water activity during sporogenesis on the germination of P. chrysogenum was greater at 0.95 aw ge than at 0.99 aw ge (Judet et al., 2008). Therefore, interaction between the water activity during sporogenesis and during germination probably occurred in the present study. Accordingly, the proposed model cannot be used for predicting the germination time in other experimental conditions than those described here. In order to quantify the combined effects, a full factorial matrix should be used. Four other experiments should be carried out in addition to the four experiments already described in this study. As compared to conidia produced at 0.99 aw sp, the germination time of all the species except P. expansum was shortened for conidia produced at 0.95 aw sp. It was suggested that the enhancement of the germination of conidia obtained under osmotic stress may be due to a greater gradient of water across the cell membrane (Judet et al., 2008). Dormant spores are characterized by a low hydration and prior to germination most species require the presence of liquid water or high relative humidity. During activation, large amounts of free water will enter into the spore, rapidly at first, leading to spore swelling (Isaac, 1998). The enhancement of conidial germination due to synthesis and/or accumulation of glycerol and erythritol were shown for several species under NaCl-induced osmotic stress (Hallsworth and Magan, 1995) and for A. nidulans under ethanol and NaCl-induced osmotic stress (Hallsworth et al., 2003). This study suggested that the water activity of suspension had a greater effect on germination time than did the kind of mould. Moulds
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