Influence of temperature, copper and CO₂ on spore counts and geosmin production by Penicillium expansum

Abstract

Background and Aim

Musts and wines produced from rotten grapes often have an earthy/musty odour, with geosmin the responsible compound. Penicillium expansum is considered a potential source of geosmin in rotten grapes from vineyards treated with copper-based fungicides.

Methods and Results

The laboratory study assessed the influence of temperature (10–30°C), copper concentration (0–76.50 mg/L) and CO₂ in the headspace (0.03–3%) on the spore count and the production of geosmin by P. expansum according to a Doehlert design. The spore count and the production of geosmin (ng/mg biomass) were significantly correlated (r = 0.78). Copper had no significant effect on the spore count but was the most important factor for explaining geosmin production. The production of geosmin was enhanced at low temperature (15°C), 0.03% CO₂ (i.e. atmospheric level) and high copper concentration (76.50 mg/L).

Conclusion

Penicillium expansum, grown on Czapek agar, produced a significant amount of geosmin at low temperature and in the presence of copper.

Significance of the Study

This study suggests a possible explanation for the occurrence of earthy/musty odours in musts and wines made from rotten grapes.

Introduction

Grape rot is one of the major causes of degradation of grape compounds leading to deterioration of wine quality. Over the past 10 years, winegrowers in Burgundy have observed sensory defects with earthy/musty odours in musts and wines made with rotten grapes. The compound responsible for this odour in Bordeaux wines was identified as geosmin (Darriet et al. 2000). The content of geosmin in various Bordeaux wines varied between 20 and 300 ng/L, thus indicating that the concentration was sometimes clearly higher than the perception threshold of racemic mixture of geosmin in water, c. 20 ng/L (Maga 1987).

Penicillium expansum and other species have long been associated with distinctive odours. Raper and Thom (1949) described the odour of various penicillia as being ‘strong, moldy or earthy’ (P. expansum and P. crustosum Thom), or ‘mouldy’ (P. claviforme Bain and P. olivino-viride Biourge). Mattheis and Roberts (1992) identified geosmin as the primary component of the odour associated with P. expansum. Microbiota on grapes with earthy odour was analysed, and its potential to produce geosmin on malt agar and grape juice was tested. According to La Guerche et al. (2004, 2006), all the strains producing geosmin belong to only one Penicillium species: P. expansum. Some species of Botrytis cinerea can induce the production of geosmin by P. expansum on grapes (La Guerche et al. 2005, Morales-Valle et al. 2011).

Geosmin production is affected by environmental and nutritional factors. On malt agar medium, temperature, humidity and oxygen concentration affected growth, sporulation and geosmin production of P. expansum but did not have a direct effect on its synthesis (La Guerche et al. 2005). A study of the impact of grape juice composition on geosmin production by P. expansum revealed the importance of nitrogen composition (La Guerche and De Seneville 2007). Ammonium played a lead role in inducing geosmin synthesis. In contrast, mixed amino acids inhibited geosmin synthesis.

Global warming may lead to important variations in rainfall, temperature and the proportion of carbon dioxide in the atmosphere. An increased accumulation of geosmin in P. expansum grown at 40°C was exhibited. No geosmin, however, was produced 2 days after inoculation of the mould on Czapek agar, at a temperature in the range 10–30°C (Dionigi and Ingram 1994). An increase in geosmin accumulation was also reported by these authors when P. expansum was incubated at 10% O₂. But, to our knowledge, the effect of CO₂ on geosmin accumulation in P. expansum has never been studied. As compared with the proportion of CO₂ in the atmosphere (0.03%), the upper value of the experimental domain was arbitrarily fixed at 3% to highlight a possible effect of this factor.

Copper salts in the form of Bordeaux mixture (Ca(OH)₂ + CuSO₄) are widely applied as a fungicide against mildew of the grapevine. An increased growth and geosmin accumulation in P. expansum were observed through the addition of copper (1 and 5 mg/L) to growth media (Dionigi 1995, Dionigi et al. 1996). More recently, it was shown that geosmin accumulation was enhanced at 1 and 1.2 mmol/L (i.e. 76.50 mg/L) copper for two strains of P. expansum (Charpentier et al. 2011). In contrast, no geosmin was produced by these strains at 0.2 mmol/L. No information, however, was available for traces of copper in the range 0.2–1.2 mmol/L. This study assessed the combined effect of temperature (10–30°C), copper concentration (0–76.50 mg/L) and CO₂ in the headspace (0.03–3%) on the production of spores and geosmin by P. expansum according to a Doehlert design.

Materials and methods

Isolation of microorganisms

Microorganisms were isolated from rotten Vitis vinifera cv. Pinot Noir, collected in Morey-Saint-Denis vineyards in 2007. Each sample was washed with a solution (1 mL/g of grape) of saline water (NaCl 9 g/L) containing Tween 80 (0.1% v/v) to remove microorganisms from the berries. The suspensions were stirred for 1 h at ambient temperature (approximately 20°C). Fractions of 0.1 mL of the diluted suspensions (from 10⁻¹ to 10⁻⁸) were spread on Petri dishes containing Rose Bengal agar medium to inhibit bacterial growth and to reduce development of Mucorales, and incubated at 25°C. After 7 days incubation, colonies were counted, isolated in pure culture and maintained on potato dextrose agar (BioMérieux, Marcy l'Etoile, France). Among the isolated strains, P. expansum was identified at the laboratory according to macroscopic criteria (Samson et al. 2004). The identification was further confirmed by a molecular approach based on the amplification of the β-tubulin gene by Bt2a and Bt2b primers (Judet-Correia 2011).

Medium and incubation devices

Czapek broth (Difco, Pessac, France) solidified by agar (15 g/L) was the basal medium (Raper and Thom 1949, Dionigi and Ingram 1994). Cultures of P. expansum were incubated in the range 10–30°C. The effect of CO₂ was investigated in a water-jacketed CO₂ incubator (Forma Series II 3110, Thermo Scientific, Courtaboeuf, France) connected to a cryostat (Minichiller Huber, VWR, Fontenay sous Bois, France) with atmospheres containing 0.03, 1.5 and 3.0% CO₂. The medium was supplemented with copper sulfate (CuSO₄.5H₂0) to give a range from 0 to 76.50 mg/L copper. Petri dishes were incubated for 7 days in hermetically closed devices that contained water to maintain a constant relative humidity throughout the experiments, as described previously (Sautour et al. 2001a). Three Petri dishes were used per experimental condition to determine the spore counts, the biomass and geosmin. All raw data were expressed per Petri dish. Geosmin was analysed only once; biomass and spore counts were determined three times, but the mean only was used.

Experimental matrix

An experimental domain was defined over 10–30°C, copper (0–76.50 mg/L) and CO₂ (0.03–3.0%). The Doehlert design allows the description of a region around an optimal response and contains k² + k + 1 points for k factors. For three factors, a set of 13 experiments was required, and in that case, one of the properties of the Doehlert design is the uniform distribution of the experiments in a three-dimensional space (Sautour et al. 2001b). Thus, 12 experiments are equidistant from a central experiment (Experiment 13) having the coded values (0, 0, 0). Experimental values are listed in Table 1. As compared with the central composite design, one of the advantages of the Doehlert design is to select a different number of levels for each factor. In the present study, three, five and seven levels were attributed to CO₂, temperature and copper, respectively.

Table 1. Experimental values for assessing the influence of factors (temperature, copper concentration and CO₂) on the production of spores and geosmin by Penicillium expansum on Czapek agar

Experiment	Coded values			Experimental values			Responses
	X1	X2	X3	T °C	Copper (mg/L)	CO2 (%)	Spores × 10^−8	Geosmin (ng/mg biomass)
^a Raw data, geosmin (ng) and biomass (mg) in italics. X1, temperature; X2, copper; X3, CO2.
1	+1	0	0	30	38.25	1.5	0.05	0.180 (5/28.2)a
2	−1	0	0	10	38.25	1.5	2.55	104 (710/6.81)
3	+0.5	+0.866	0	25	76.50	1.5	0.70	47.4 (1659/35.0)
4	−0.5	−0.866	0	15	0.00	1.5	1.60	7.90 (84/10.7)
5	+0.5	−0.866	0	25	0.00	1.5	1.25	0.630 (17/26.8)
6	−0.5	+0.866	0	15	76.50	1.5	2.80	267 (2650/9.82)
7	+0.5	+0.289	+0.816	25	51.00	3.0	0.35	0.180 (5/28.2)
8	−0.5	−0.289	−0.816	15	25.50	0.03	3.40	114 (1939/17.1)
9	+0.5	−0.289	−0.816	25	25.50	0.03	2.10	15.9 (300/18.8)
10	0	+0.577	−0.816	20	63.75	0.03	4.65	196 (3150/16.1)
11	−0.5	+0.289	+0.816	15	51.00	3.0	1.05	1.04 (13/12.5)
12	0	−0.577	+0.816	20	12.75	3.0	0.20	0.620 (5/8.12)
13	0	0	0	20	38.25	1.5	0.70	0.620 (5/8.12)

Spore count

A colony from a Petri dish was transferred to 100 mL of sterile water supplemented with Tween 80 (0.1% v/v) and 20 glass beads (2-mm diameter) contained in a bottle stirred at 110 rpm for 30 min to detach the spores. Spores were enumerated by means of a haemocytometer.

Biomass

The agar was cut around the colony and transferred to 100 ml of sterile water contained in a bottle which was placed into a microwave at 650W for 90–120 s depending on the size of the colony. The bottle was gently stirred by hand for 1–2 min to dissolve the agar into the water. The colony was then removed from the bottle and placed on a pre-weighed Whatman paper. The colony was washed two to three times with 3 mL of boiling water. The paper and the colony (i.e. sample) were placed in a glass Petri dish and dried for 24 h at 86°C in an oven. The sample was then placed into a dryer for 1 h and weighed. The operation was repeated until a constant weight.

Geosmin

The sporulating mycelium and the agar medium (approximately 15 mL) were removed from the plate and mixed for 1 h at 20°C into 100 mL of aqueous ethanol (20% v/v) with a magnetic stirrer at 250 rpm. After filtering the mixture, a 6-mL aliquot of sample was diluted with ultra pure water PURELAB UHQ (ELGA LabWater, Antony, France). Geosmin was quantified at Laboratoire Exact (Macon, France) following the protocol described previously (Dumoulin and Riboulet 2004). Briefly, geosmin (d5) was used as an internal standard. Solutions of geosmin and geosmin (d5) were prepared by dilution with absolute ethanol. The volatile compounds were extracted from the sample by solid-phase microextraction (SPME) on a polydimethylsiloxane fibre. The molecules were desorbed from the SPME fibre, and the sample was injected into a gas chromatograph coupled to a mass spectrometer ion trap. The volatile compounds were separated on a capillary column. Geosmin and geosmin (d5) were fragmented twice using the mass spectrometry technique into the ions 112 then 97, and into the ions 114 then 99, respectively. Quantification was based on the ions 97 (geosmin) and 99 (geosmin d5). The limit of detection and of quantification of geosmin (5 and 10 ng/L, respectively) was similar to that reported by Morales-Valle et al. (2010). The repeatability was 6.7% at 10 ng/L and 6.1% at 100 ng/L.

Analysis and interpretation of the results

The correlation between the experimental responses was assessed with Student's t-test, as described previously (Dantigny et al. 2005). Spore counts and geosmin (ng/mg biomass) were modelled by a second-order polynomial relationship that included quadratic terms.

• (1)

where i = spores count and geosmin; and X₁ (temperature), X₂ (copper) and X₃ (CO₂) = coded factors studied.

The linear and quadratic effects of the three factors and their interactions were subjected to multiple regression analysis based on the least square method (Nemrodw software, LPRAI, Marseille, France). The significance of the coefficients was evaluated by multiple regression analysis based upon the F-test with unequal variance, P < 0.05 (*) and P < 0.01 (**).

Results and discussion

The raw data – geosmin (ng), biomass (mg) and spore counts (×10⁻⁸) – are shown in Table 1. No correlation was found between biomass and the spore counts (r = −0.31). In contrast, a significant correlation (r = 0.78) was shown between geosmin (ng/mg biomass) and the spore counts (Figure 1). This result was in accordance with the observations of La Guerche (2004) on two strains of P. expansum growing on Czapek agar or malt agar media. In that study, the maximal level of geosmin (13 and 14 μg/g biomass) was obtained when sporulation occurred. Most secondary metabolites are produced in the centre of the colony where the formation of spores occurs. For this reason, a correlation was found in the present study between the spore count and geosmin production. This correlation between spore formation and secondary metabolite production has been corroborated by Calvo et al. (2002) who showed that both conidiation and sterigmatocystin biosynthesis by Aspergillus nidulans are co-regulated by a common signal transduction pathway. At present, there is no evidence that such a phenomenon could explain the correlation between conidiation and geosmin production by P. expansum.

Figure 1. Correlation between the spore count of Penicillium expansum grown on Czapek agar for 7 days and the production of geosmin (ng/mg biomass).

Spore count and geosmin production are correlated positively (r > 0) when more than about 20 ng geosmin/mg biomass were produced (Figure 1). As such, it was shown that the experimental conditions that maximised the spore counts (X₁ = −0.43, X₂ = 0.39 and X₃ = −0.79) were similar (i.e. the signs of the factors were identical) to those that maximised the production of geosmin (X₁ = −0.52, X₂ = 0.65 and X₃ = −0.43) (Figures 2, 3). In contrast, when the correlation was less obvious (i.e. at low spore count), the experimental conditions that minimised the spore count (X₁ = 0.79, X₂ = 0.32 and X₃ = 0.43) differed from those that minimised the production of geosmin (X₁ = −0.43, X₂ = −0.36 and X₃ = 0.61).

Figure 2. Process optimisation exhibiting experimental conditions that minimised and maximised production of spores by Penicillium expansum. X₁, temperature; X₂, copper; X₃, CO₂.

Figure 3. Process optimisation exhibiting experimental conditions that minimised and maximised geosmin production by Penicillium expansum. X₁, temperature; X₂, copper; X₃, CO₂.

The model coefficients are listed in Table 2. The major effect of temperature and CO₂ was significant for explaining the spore count, whereas no interactive effect was significant. The Pareto chart demonstrated the paramount effect of CO₂ on the spore count (Figure 4). Because of a negative b₃ value, the spore count was increased for a negative value of X₃. Conversely, an elevated carbon dioxide proportion decreased the spore count. This effect was also shown for the conidiation in Neurospora crassa strains (Sargent and Kaltenborn 1972). Similarly, because of a negative b₁ value, the spore count was increased for a negative value of X₁ (i.e. temperature less than 15°C). It was shown (experiment 1) that almost no spores of P. expansum were produced at 30°C. In fact, the optimum temperature for growth is slightly higher than that for spore production (Muller 1956). Spore production of Metarhizium anisopliae was greater at 20°C than at 15 and 30°C (El Damir 2006). As compared with a maximum temperature for growth in the range 32–37°C (Pitt and Hocking 2009), the maximum sporulation by Fusarium verticillioides occurred at 27°C, then a rapid decline was observed, with no sporulation at 45°C (Rossi et al. 2009).

Figure 4. Pareto chart assessing the linear, quadratic and interactive effects of temperature, copper and CO₂ on the production of spores by Penicillium expansum.

Table 2. List of model coefficients and P-values

Coefficient	Spores ×10^−8		Geosmin
	Value	P-value	Value	P-value
*P < 0.05; **P < 0.01
b0 response means	0.700	33.6	0.620	98.3
b1 T	−1.182	2.90*	−66.71	2.30*
b2 copper	0.487	20.9	85.17	1.11*
b3 CO2	−1.746	0.916**	−66.13	2.36*
b11 T^2	0.600	48.4	51.62	27.7
b22 copper^2	0.984	28.1	89.58	10.4
b33 CO2^2	1.492	13.2	45.68	31.4
b12 T × copper	−1.011	24.8	−122.5	4.29*
b13 T × CO2	0.725	42.8	102.7	8.6
b23 copper × CO2	−0.855	35.9	−79.49	14.7

The effect of the experimental factors on geosmin production was more difficult to interpret because of the importance of the interactive effects, as described by the Pareto chart (Figure 5). Contour plots were drawn to analyse the experimental conditions that maximised the production of geosmin. The combined effect of temperature and copper on geosmin production is shown in Figure 6. Independent of the copper concentration, the production of geosmin was negligible at 30°C. In contrast, geosmin production was greater at 10°C than at 30°C. Saadoun et al. (2001) analysed the geosmin production by the cyanobacteria Anabaena sp. They observed that the ratio of the geosmin produced over the biomass was maximum at 15°C, whereas the geosmin production greatly decreased above 20°C. At 10°C, the production of geosmin by P. expansum was greatly affected by the copper concentration. A geosmin concentration greater than 250 ng/mg of biomass was obtained for a copper concentration in the range 38.25–76.50 mg/L.

Figure 5. Pareto chart assessing the linear, quadratic and interactive effects of temperature, copper and CO₂ on the production of geosmin by Penicillium expansum.

Figure 6. Contour plot of the influence of copper and temperature on the production of geosmin (ng/mg biomass) on Czapek agar by Penicillium expansum at 0.855% CO₂.

The greater production of geosmin at 10°C than at 30°C is also shown in Figure 7. It should be emphasised that no geosmin was produced at 30°C, even at a copper concentration, 63.10 mg/L that favoured geosmin production. At 10°C, geosmin production was negatively affected by an increase of CO₂ concentration. Similarly, the negative effect of a high carbon dioxide concentration is shown in Figure 8, especially at 76.50 mg/L copper. It was shown that an elevated carbon dioxide concentration decreased the production of geosmin in Streptomyces albidoflavus (Sunesson et al. 1997). The decrease in the production of secondary metabolites at an elevated CO₂ concentration was observed for other fungi. At 20% CO₂ concentration, a low biomass and penicillin concentration were reported by Ho and Smith (1986), thus indicating an inhibition of both primary and secondary metabolism. At least 50% CO₂ was needed to inhibit P. verrucosum growth and ochratoxin A production by 75% (Cairns-Fuller et al. 2005).

Figure 7. Contour plot of the influence of CO₂ and temperature on the production of geosmin (ng/mg biomass) on Czapek agar by Penicillium expansum at 63.10 mg/L copper.

Figure 8. Contour plot of the influence of CO₂ and copper on the production of geosmin (ng/mg biomass) on Czapek agar by Penicillium expansum at 14.8°C.

Conclusions

Up to 267 ng geosmin per mg biomass were produced on Czapek agar after 7 days incubation at 15°C and 76.50 mg/L copper under atmospheric CO₂. But, a significant geosmin concentration can also be obtained in less favourable conditions under atmospheric CO₂. For example, 196 ng geosmin/mg biomass were produced at 20°C and 63.75 mg/L copper. It is difficult to correlate the concentration of geosmin obtained in this study with the concentration that can be found in wine. But, because of a low perception threshold, earthy/musty odours may be detected in wines for environmental conditions far from optimum. The enhancement of geosmin production in P. expansum grown on Czapek agar was demonstrated at low temperature, high copper content and atmospheric CO₂ concentration. This study carried out in the laboratory suggests a possible explanation for the occurrence of earthy/musty odours in musts and wines made from rotten grapes. But, more studies should be conducted in the vineyards to assess the effect of copper on the production of geosmin in P. expansum on grapes.

Acknowledgements

Authors wish to thank the National Program on earthy/musty taste of vine (FRANCE AGRIMER, Paris). Marie-Christine Guilhem from LPRAI is gratefully acknowledged for her helpful comments.