Intraspecific variability of growth and ochratoxin A production by Aspergillus carbonarius from different foods and geographical areas M.R. Bragulat, M.L. Abarca, G. Castellá*, F.J. Cabañes Veterinary Mycology Group, Department of Animal Health and Anatomy, Universitat Autònoma de Barcelona, Bellaterra, Spain. *Corresponding author: Gemma Castellá Grup de Micologia Veterinària Departament de Sanitat i d'Anatomia Animals Facultat de Veterinària, Universitat Autònoma de Barcelona 08193 Bellaterra (Barcelona), Spain. Tel: +34 93 5811089 Fax: +34 93 5812006 e-mail: Gemma.Castella@uab.cat 1 1 Abstract 2 Ochratoxin A (OTA) is a nephrotoxic mycotoxin naturally found in a wide range of 3 food commodities throughout the world. Aspergillus carbonarius is the most important 4 source of OTA in food commodities such as wine, grapes and dried vine fruits and is 5 also responsible for the formation of OTA in coffee. 6 The aim of this study was to determine the simultaneous effect of three culture 7 media (Czapek Yeast Extract Broth (CYB); Synthetic Grape Juice Medium (SGM) and 8 White grape juice (WGJ)) at three water activity (aw) levels (0.90; 0.95 and 0.98-0.99), 9 and three incubation temperatures (15ºC, 25ºC and 35ºC) on the growth and OTA 10 production by 16 strains of A. carbonarius. The strains were selected on the basis of 11 the geographical origin of the substrate and included strains from different climatic 12 zones of Spain as well as from other countries with different climatology. All the strains 13 were confirmed for identity by sequencing of the calmodulin gene. The assay was 14 performed in microtiter plates, determining the absorbance at 530 nm and the 15 concentration of OTA after 1, 2, 4 and 10 days of incubation. 16 No significant differences were observed in absorbance values between the 17 strains. The highest absorbance values were recorded in CYB at 0.99 aw and at 0.95 aw 18 after 10 days of incubation at 25ºC and 35ºC. None of the strains were able to grow at 19 0.90 aw and 15ºC in any culture media after 10 days of incubation. OTA concentration 20 was statistically higher at 15ºC than at 25ºC or 35ºC. The highest significant OTA values 21 were obtained at 0.98-0.99 aw and the best culture media for OTA production was CYB, 22 followed by WGJ and SGM. While strains isolated from Mediterranean climate foods 23 had a similar behavior despite being isolated from different geographical areas, OTA 2 24 concentration produced by one Robusta coffee strain from Thailand was statistically 25 higher at 25ºC than at 15ºC. This would suggest that the type of food matrices and 26 consequently the adaptation of A. carbonarius strains to different climatic conditions 27 would have a greater influence on the ecophysiology of the strains than only their 28 geographical origin. 29 30 Keywords: Aspergillus carbonarius, coffee, ecophysiology, grapes, raisins, ochratoxin A 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 3 48 1. Introduction 49 50 Ochratoxin A (OTA) is a mycotoxin produced by several fungal species of the 51 genera Penicillium and Aspergillus. OTA is nephrotoxic, hepatotoxic, neurotoxic, 52 teratogenic and immunotoxic in various animals in vitro, with renal toxicity and 53 carcinogenesis being the key adverse effects (Heussner and Bingle, 2015). OTA 54 contamination of food commodities, including cereals and cereal products, pulses, 55 coffee, beer, grape juice, dry vine fruits and wine as well as cacao products, nuts and 56 spices, has been reported from all over the world (EFSA 2006). In the European diet, 57 wine was identified as the second contributor to the human OTA exposure after 58 cereals. Dried vine fruit and grape juice contributed to a significant extent to the OTA- 59 exposure for specific groups of vulnerable groups of consumers such as children. 60 Coffee represented the third contributor to the mean European total dietary intake of 61 OTA (European Commission, 2002). As a consequence of the possible health hazards 62 related to ingestion of OTA, the European Union introduced maximum limits for OTA in 63 a wide range of foodstuffs (Commission of the European Communities 2006; 2010; 64 2012; 2015). 65 A large number of studies have shown that Aspergillus carbonarius is the main 66 responsible source of OTA in wine or dried vine fruits from main vinicultural regions 67 worldwide (Cabañes and Bragulat 2018; Visconti et al.,2008). According to recent 68 surveys, A. carbonarius is also the most important ochratoxin producer in Robusta 69 coffee beans (Noonim et al., 2008). 70 Aspergillus carbonarius is very consistent in producing this mycotoxin, and non 71 OTA-producing strains in this species are very rare (Cabañes et al., 2013). 4 72 Contamination of grapes by this species can occur since the beginning of maturation 73 stage but becomes more prominent near harvest time (Bau et al., 2005; Visconti et al., 74 2008). With respect to coffee, OTA contamination can be regarded as a post-harvest 75 problem. The use of dry processing method, surface contact with dust and soil increase 76 the chance of A. carbonarius contamination (Noonim et al., 2008). 77 The knowledge of the influence of environmental parameters on OTA production 78 may contribute to prevention of OTA contamination in food commodities. In the last 79 years many studies have been published on the effect of some environmental factors 80 on A. carbonarius growth and in a lesser extent on its OTA production ability, as 81 reviewed by several authors (Amézqueta et al., 2012; Battilani and Camardo, 2015; 82 Magan et al., 2011). These kind of ecophysiological studies are routinely performed in 83 Petri dishes and the number of isolates included is usually low because they are 84 laborious and time-consuming methods. 85 In order to know the existence of intraspecific variability on growth and OTA 86 production, several strains have to be included in ecophysiological studies. Garcia et al. 87 (2011) assessed the impact of suboptimal environmental conditions on thirty isolates 88 of A. carbonarius. Although they included a large number of isolates, only one culture 89 medium and three water activity/temperature conditions (0.98 aw/25ºC; 0.90 90 aw/25ºC; 0.98 aw/37ºC) were tested. Using above data, García et al. (2012) 91 mathematically assessed the minimum number of isolates that would lead to 92 equivalent growth parameters estimates to those obtained with a high number of 93 strains. They concluded that 12-17 isolates of A. carbonarius led to the same growth 94 parameters as the total 30. 5 95 In this study we have adapted a previously described method using microtiter 96 plates (Abarca et al., 2014; 2019) to determine the simultaneous effect of three culture 97 media at three water activity levels and three incubation temperatures on the growth 98 and OTA production by 16 strains of A. carbonarius. 99 100 2. Materials and Methods 101 102 2.1. Strains and molecular identification 103 104 Sixteen A. carbonarius strains, mainly isolated from grapes and raisins were 105 studied (Table 1). All the strains were previously detected as OTA-producers in our 106 laboratory and had been morphologically identified as A. carbonarius. The strains were 107 selected on the basis of the geographical origin of the substrate and included strains 108 from different climatic zones of Spain as well as from other countries with different 109 climatology. 110 All the strains were confirmed for identity by sequencing of the calmodulin gene. 111 Briefly, DNA was extracted and purified from 48 h old cultures in malt extract broth 112 according to the FastDNA Spin kit protocol with the FastPrep FP-24 instrument (MP 113 Biomedicals, Biolink, Barcelona, Spain). The DNA was kept at -20 ºC until used as 114 template for PCR amplification. Following the DNA extraction, the calmodulin gene 115 was amplified and sequenced by using the fungal primers CL1/CL2A (O’Donnell et al., 116 2000). For the phylogenetic analyses, sequences obtained were aligned using Clustal X 117 v2.0.12 (Larkin et al., 2007) and analyzed to generate a phylogenetic tree in Mega 6 6 118 software (Tamura et al., 2013). The Neighbor-Joining method based on the Tamura- 119 Nei model (Tamura and Nei 1993) with 1,000 bootstrap replicates was used. 120 121 2.2. Inoculum preparation and verification 122 123 Spore concentration was adjusted to around 106 conidia/ml. Briefly, the inoculum 124 suspensions were prepared in sterile saline (0.85%) containing 0.05% Tween 80 from 125 7-day-old cultures on malt extract agar at 25ºC. After heavy particles were allowed to 126 settle for 10-15 minutes, the upper homogenous suspensions were transferred to 127 sterile tubes and adjusted to 0.8 McFarland turbidity standard (Abarca et al., 2014) by 128 using a photometric method (Densimat, BioMérieux). The inoculum size was confirmed 129 by haemocytometer counting and quantitative colony counts. 130 131 2.3. Culture media and microtiter inoculation 132 133 Sterile 96-well flat-bottom microtiter plates were used. Three liquid culture media 134 were assayed: Czapek Yeast Extract broth (CYB), used as a control, synthetic grape 135 juice medium (SGM) representative of grape composition at mid-veraison (Mitchell et 136 al., 2004), and white grape juice (WGJ). 137 CYB contained per liter: K2 HPO4, 1 g; Czapek concentrate, 10 ml; trace metal 138 solution, 1 ml; yeast extract, 5 g; sucrose, 30 g; pH adjusted to 6.3 ± 0.2 (Pitt and 139 Hocking, 2009). SGM contained per liter: D(+) glucose, 70g; D(-) fructose, 30g; L(-) 140 tartaric acid, 7g; L(-) malic acid, 10g; (NH4)2HPO4, 0.67g; KH2PO4, 0.67g; MgSO4·7 H2O, 141 1.5g; NaCl, 0.15g; CaCl2, 0.15g; CuCl2, 0.0015g; FeSO4· 7 H2O, 0.021g; ZnSO4·7 H2O, 7 142 0.0075g; (+) Catechin hydrate, 0.05g; pH adjusted with 10M NaOH to pH 4.0-4.2 143 (Mitchell et al., 2004). WGJ was prepared with 200ml of commercially sold white grape 144 juice made from ecological grapes and 800 ml of distilled water; pH adjusted at 4.0. 145 The initial aw was 0.99 for CYB and WGJ media, and 0.98 for SGM. These initial 146 values were modified to 0.95 aw and 0.90 aw by the addition of different amounts of 147 glycerol. Media were autoclaved and the final aw values were checked with 148 LabMASTER-aw (Novasina. Switzerland). 149 For each aw level the adjusted fungal suspensions were diluted 1:100 in the culture 150 medium assayed (CYB, SGM, WGJ). In each microplate column, five wells were 151 inoculated with 200 µl of the diluted suspension of each strain and one well, used as a 152 blank, was filled with 200 µl of un-inoculated culture media (CYB, SGM ,WGJ). 153 Growth assessment and OTA production at each aw level were determined after 1, 154 2, 4, and 10 days of incubation at three different temperatures (15, 25, and 35ºC). 155 Thus, each strain-awlevel-temperature combination was repeated in 4 microplates, one 156 for each sampling day. 157 For each sampling occasion and temperature assayed, microtiter plates with the 158 same water activity level were enclosed in sealed polyethylene bags. The entire 159 experiment was repeated twice on different days. 160 161 2.4. Growth measurement and OTA extraction procedure 162 163 For each culture media, aw level and temperature assayed, growth was monitored 164 by absorbance measurements at 530nm using the Multilabel-Reader Mithras LB 940 165 (Berthold Technologies, Bad Wildbad, Germany) after 1, 2, 4 and 10 days of 8 166 incubation. The absorbance of the corresponding uninoculated medium, used as blank 167 was subtracted to the absorbance values of the inoculated media. After each reading, 168 microplates were sealed and stored at -80ºC until they were analyzed for OTA 169 content. 170 OTA production was detected using a previously described high-pressure liquid 171 chromatography (HPLC) screening method developed in our laboratory for fungi 172 growing in microtiter wells (Abarca et al., 2014). On each sampling occasion, one of the 173 five replicate wells inoculated for each strain, culture media, aw level and incubation 174 temperature, were randomly selected and their content was removed and extracted 175 with 0.5 ml of methanol. The extracts were filtered and injected into the HPLC. The 176 limit of quantification was 0.045 µg/ml for this mycotoxin. 177 178 2.5. Statistical Analysis 179 180 Data obtained from the different conditions tested were statistically analyzed by 181 means of one-way analysis of variance test. All statistical analyses were performed 182 using Minitab 17 statistical software (Minitab Inc., State College, Pennsylvania, USA). 183 184 3. Results 185 186 3.1. Molecular species identification 187 188 Based on the calmodulin sequences, all the strains were identified as A. 189 carbonarius. The phylogenetic tree was reconstructed showing that the isolates 9 190 grouped with A. carbonarius CBS 556.65T (Fig. 1). Sequence analysis revealed that all 191 the strains had the same sequence. The nucleotide sequence of one representative 192 strain (A-1002) has been deposited in GenBank under accession number MK778845. 193 194 3.2. Inoculum standardization 195 196 Inocula adjusted to 0.8 McFarland turbidity standard provided suspensions of 1.1 ± 197 0.1 x 106 conidia/ml by microscopic enumeration with a cell-counting 198 haemocytometer. Mean colony counts of the above A. carbonarius suspensions were 199 0.3 ± 0.1 x 106 cfu/ml. 200 201 3.3. Growth measurement 202 203 Table 2 shows results of one-way analysis of variance for the effects of the 204 variables assayed (experiment, temperature, water activity, culture media and strain) 205 on absorbance values. No significant differences were observed in absorbance values 206 neither between the experiments, nor between the strains (p > 0.05). The remaining 207 variables had a significant effect on growth of all the strains studied. Regardless aw 208 level or culture medium, the highest absorbance values were recorded at 35ºC, 209 although not statistically different from those obtained at 25ºC. Values recorded at 210 0.98-0.99 aw and 0.95 aw were significantly higher than at 0.90 aw. In relation to culture 211 media, the highest significant absorbance values (p<0.001) were recorded in CYB, 212 followed by SGM and WGJ. 10 213 Mean absorbance values recorded in both experiments by all the studied 214 strains at each condition and incubation time tested are shown in Table 3. The highest 215 absorbance values were recorded after 10 days of incubation at 25ºC and 35ºC in CYB 216 at 0.99 aw and at 0.95 aw. 217 In all culture media, initial growth in the microtiter wells could be visually detected 218 at the naked eye, when absorbance value was greater than 0.1. According to this, none 219 of the strains were able to grow at 0.90 aw and 15ºC, the most extreme conditions 220 tested, in any culture media after 10 days of incubation. At this low temperature, a 221 significant increase in absorbance values was detected after 4-10 days of incubation in 222 the three culture media adjusted at 0.95 and 0.98-0.99 aw. 223 At 25ºC, absorbance values statistically increased after 2-4 days (0.98-0.99 aw and 224 0.95 aw) or 10 days (0.90 aw) of incubation. 225 At 35ºC a statistically growth increase was observed after 2 days of incubation in 226 the three culture media adjusted at 0.98-0.99 aw and 0.95 aw. At 0.90 aw, this increase 227 was recorded after 2-4 days in SGM and WGJ and after 10 days in CYB. 228 229 3.4. OTA production 230 231 No significant differences were found in OTA concentration neither between the 232 experiments, nor between the strains (p > 0.05) (Table 2). Temperature, culture media 233 and water activity had significant effect (p<0.01) on OTA production. OTA 234 concentration was statistically higher (p<0.01) at 15ºC than at 25ºC or 35ºC. The 235 highest significant OTA values were obtained at 0.98-0.99 aw and the best culture 236 media (p<0.01) for OTA production was CYB, followed by WGJ and SGM. 11 237 Table 4 shows OTA concentration produced by all the strains at each condition 238 assayed and incubation time. Results are expressed as mean value of both experiments 239 as no statistically significant differences were observed (p>0.05). As a general trend, 240 OTA production increased over incubation time achieving the highest concentration 241 (p<0.01) after 10 days of incubation. None of the strains produced detectable levels of 242 OTA in any culture media at 0.90 aw and 15ºC. The highest OTA concentration was 243 recorded at 15ºC after 10 days of incubation in CYB at 0.99 aw.. It is worth to highlight 244 that in those conditions of temperature and aw, all the strains produced detectable 245 levels of OTA in the three culture media studied. 246 At 25ºC and 0.98-0.99 aw , all the strains produced OTA after only 2 days (CYB, 247 WGJ) or 4 days (SGM) of incubation. As water activity decreases, optimum 248 temperature for OTA production moved to 25ºC, although not all the studied strains 249 were able to produce the mycotoxin. 250 At 35ºC, and 0.98-0.99 aw some strains produced low levels of OTA after only 1 or 251 2 days of incubation. By decreasing the water activity, decreases the number of OTA- 252 producing strains. At 0.90 aw, still some strains could produce detectable levels of OTA 253 after 10 days of incubation. 254 Although taking into account all the results no intraspecific differences were 255 observed, the strain A-884 isolated from Robusta coffee cherries from Thailand, 256 showed a different behavior to the remaining strains, mostly isolated from grapes. 257 Thus, OTA concentration produced by this strain was statistically higher (p<0.05) at 258 25ºC than at 15ºC or 35ºC. Fig. 2a shows OTA values recorded in CYB medium by this 259 coffee strain (A-884) in comparison to a high OTA- producing grape strain (A-1002) and 260 a low OTA-producing grape strain (A-2128), both isolated from Spain, in each condition 12 261 assayed. The monthly maximum and minimum average temperatures in 262 Mediterranean areas and in Thailand are also shown (Fig. 2b). 263 264 4. Discussion 265 For each strain and reading day (1, 2, 4 and 10 days), the total number of 266 conditions studied were 27 (3 culture media at 3 water activities and 3 incubation 267 temperatures). The large number of isolates and conditions studied here will allow a 268 more comprehensive overview of the ecophysiology of A. carbonarius, difficult when 269 using laborious methods based on Petri dishes. 270 Inoculum size is one of the technical variables that can influence the 271 spectrophotometric tests outcome. Conidial suspensions adjusted to 0.8 McFarland 272 yielded in all cases haemocytometer counting around 106 conidia/ml. The use of the 273 portable photometer Densimat (BioMérieux) provides a substantial reduction in time 274 as we have previously reported (Abarca et al., 2014; 2019; Cabañas et al., 2009). As no 275 statistically significant differences were observed in absorbance and OTA values 276 between the experiments, our method has proven to be an easy way to monitor 277 growth and OTA production of A. carbonarius in ecophysiological studies. 278 Results obtained in this study confirm that A. carbonarius strains can grow at a 279 wide range of temperatures (15-35ºC) and water activities (0.90-0.99). The optimum 280 conditions of aw and temperature for growth were in the range 25-35ºC and 0.95-0.99 281 aw. The highest amount of OTA was obtained at 15ºC and 0.98-0.99 aw. In previous 282 studies in solid culture media, A. carbonarius strains also showed optimal conditions 283 range for OTA production narrower than that for growth when temperature, water 284 activity or pH effects were evaluated (analyzed) (Esteban et al., 2004; 2005; 2006). 13 285 In agreement with our results, 15ºC or 15-20ºC have been reported as the 286 optimum temperatures for OTA production by A. carbonarius strains in different 287 culture media (Esteban et al., 2004; Leong et al., 2006; Marin et al., 2006; Mitchell et 288 al., 2004; Passamani et al., 2014; Romero et al., 2010; Tassou et al., 2007). 289 Although all the strains were able to produce OTA in the three culture media 290 studied, the highest OTA concentration was achieved in CYB (final pH 6.3 ± 0.2). Czapek 291 yeast extract agar (CYA) has been reported as a suitable medium to detect OTA- 292 producing ability of A. carbonarius strains (Abarca et al., 2014; Bragulat et al., 2001; 293 Esteban et al., 2004). In SGM and WGJ media, with a final pH of about 4, a decrease in 294 both growth and OTA production was obtained. The acidity of the growth medium 295 could exert some influence on the growth and OTA production by A. carbonarius. In 296 previous studies, A. carbonarius isolates were able to produce OTA at a wide range of 297 pH values (2 to 10) on CYA medium. When CYA plates were incubated at 15ºC, the 298 highest OTA levels were obtained generally at a higher pH range (5 to 7) (Esteban et 299 al., 2005). In a similar way, the greatest OTA concentration was reported in 300 semisynthetic grape culture medium at 15ºC, with pH above 6.0 and aw of 0.99 301 (Passamani et al., 2014). In other studies, low pH level seemed optimal for maximum 302 OTA production (Spadaro et al., 2010), while Kapetanakou et al. (2009) reported that 303 pH (3.9-6.8) had no particular effect on OTA production by A. carbonarius. 304 In accordance with our results some studies have shown that A. carbonarius 305 strains isolated from grapes from different geographical origins have a common 306 pattern in growth and OTA production under variable conditions of temperature and 307 aw. Strains of A. carbonarius isolated from Tunisian grapes behave as those from 308 Spanish grapes (Marin et al., 2006) and as those from Australian and European grapes 14 309 as reported in the literature (Lasram et al., 2010). Leong et al. (2006) reported that 310 trends for growth and OTA production were similar among Australian isolates and 311 those from European grapes. Using a cocktail inocula of strains isolated from 312 Argentinean dried vine fruits, similar results were obtained to those reported for single 313 A. carbonarius strains from European countries, Israel, Australia and South America 314 (Romero et al., 2010). Therefore, strains isolated from grapes seem to have a similar 315 behavior despite being isolated from different geographical areas. 316 In our study, OTA concentration produced by the strain A-884 isolated from 317 Robusta coffee cherries from Thailand was statistically higher at 25ºC than at 15ºC or 318 35ºC. Most, if not all, current ecophysiological studies have been carried out with A. 319 carbonarius strains isolated from grapes and derived products. Up to date, there are 320 few data available regarding the effect of environmental factors on A. carbonarius 321 strains isolated from coffee. In a previous study, including strains from different 322 substrates, A. carbonarius CBS 127.49 isolated from a seed of coffee Arabica produced 323 more OTA at 20ºC than at 15ºC (Esteban et al., 2004). Kouadio et al. (2007) reported 324 maximum OTA production at 0.99 a w at a temperature range of 15-30ºC in one strain 325 of A. carbonarius isolated from Robusta coffee beans. On irradiated coffee cherries, 326 optimal conditions for OTA production was also reported at 25ºC and 0.99 aw (Joosten 327 et al., 2001). In the same way, on a green coffee-based medium the highest OTA 328 production was obtained at 0.99aw and 25ºC by the A. carbonarius strain studied, 329 isolated from Arabica coffee beans (Akbar, 2015). 330 The strain A-884 was isolated from the South of Thailand (Joosten et al., 2001). In 331 this tropical area the climate is hot all year round, with maximum and minimum 332 temperature ranges of 35-40ºC and 17-27ºC respectively (Fig. 2b). The remaining A. 15 333 carbonarius strains studied were isolated from areas with Mediterranean climate, with 334 relatively mild winters and warm summers but reaching minimum temperatures of 4- 335 18ºC, lower than those observed in tropical areas (Fig. 2b). This would explain that 336 15ºC be the optimum temperature for these strains, as OTA can be continuously 337 produced in the field, in spite of there are large temperature variations between day 338 and night. 339 Although very few coffee strains have been studied so far, this different pattern in 340 relation to optimal temperature for OTA production could suggest that the type of 341 food matrices and consequently the adaptation of A. carbonarius strains to different 342 climatic conditions would have a greater influence on the ecophysiology of the strains 343 than only their geographical origin. Further studies are needed including a great 344 number of coffee isolates to corroborate it. 345 346 Acknowledgements 347 348 We thank C. Gómez for her valuable technical assistance and P. Battilani, A. D. Hocking, 349 H. Joosten, A. Lebrihi, A. Lichter, E. Tjamos and A. Venancio for kindly providing us with 350 some of the strains used in this study. 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Evol. 30, 2725-2729. 480 Tassou, C.C., Natskoulis, P.I., Panagou, E.Z., Spiropoulos, A.E., Magan, N., 2007. Impact 481 of water activity and temperature on growth and Ochratoxin A production of two 482 Aspergillus carbonarius isolates from wine grapes in Greece. J. Food Prot. 70: 483 2884-2888. 484 Visconti, A., Perrone, G., Cozzi, G., Solfrizzo, M., 2008. Managing ochratoxin A risk in 485 the grape-wine food chain. Food Add. Contam. 25, 193-202. 486 487 488 489 490 491 492 493 494 495 496 22 497 Figure captions 498 Fig. 1. Phylogenetic tree of Aspergillus section Nigri inferred from Neighbor-Joining analysis of partial calmodulin gene. Bootstrap values >70% in 1,000 replications are shown at nodes. 499 500 Fig. 2. OTA production of several A. carbonarius assayed strains: A-884 (Robusta coffee 501 cherries, Thailand), A-1002 (grapes, Spain) and A-2128 (grapes, Spain) at each 502 condition assayed (a) and monthly maximum and minimum average temperatures in 503 Mediterranean areas and in Thailand (b). 23 Table 1. List of A. carbonariusa strains studied and their source and location. Strain number b Source, location A-882 tomatoes, Morocco A-884 coffee cherries, Thailand A-1002 grapes, Spain A-1004 grapes, Spain A-1021 grapes, Spain A-1625 grapes, Italy A-1653 grapes, France A-1687 grapes, Portugal A-1749 grapes, Israel A-1841 grapes, Greece A-1995 grapes, Australia A-2128 grapes, Spain A-2275 grapes, Spain A-2520 grapes, Spain A-3615 raisins, Turkey A-3905 raisins, Iran a Identification confirmed by sequencing of the calmodulin gene. b Culture Collection of the Veterinary Mycology Group, Universitat Autònoma de Barcelona, Spain. 24 Table 2. One-way analysis of variance of Absorbance (ABS) and Ochratoxin A (OTA) values versus (vs.) each of the variables assayed. p value vs. EXP vs. T (ºC) vs. aw vs. culture media vs. strain ABS 0.078 0.000 (35º a > 25º a >15º b) 0.000 (0.99 a > 0.95 a > 0.90 b) 0.000 ( CYB a>SGM b>WGJ c) 0.999 OTA 0.257 0.000 (15º a >25º b >35º b) 0.000 (0.99 a > 0.95 b > 0.90 b) 0.000 ( CYB a>WGJ b>SGM b) 0.265 a,b,c values of variables with the same superscript are not significantly different (p > 0.05). Abbreviations: EXP, experiment; T, temperature in ºC; aw , water activity; YESB, Yeast extract sucrose broth; SGM, Synthetic grape juice medium; WGJ, White grape juice. 25 Table 3. Mean absorbance (ABS) and standard deviation values in both experiments by all the studied strains of Aspergillus carbonarius at each condition and incubation time tested. ABS ±  culture days T 15ºC 25ºC 35ºC media aw 0.99-0.98 0.95 0.90 0.99-0.98 0.95 0.90 0.99-0.98 0.95 0.90 CYB 1 0.0050 ± 0.0047 a 0.0009 ± 0.0084 a - 0.00005 ± 0.0069 a 0.0467 ± 0.0203 a 0.0035 ± 0.0054 a 0.0051 ± 0.0064 a 0.0577 ± 0.0323 a 0.0506 ± 0.0357 a 0.0185 ± 0.0280 a 2 0.0106 ± 0.0075 a 0.0010 ± 0.0121 a 0.0016 ± 0.0056 b 0.7721 ± 0.3356 b 0.0455 ± 0.0181 b 0.0034 ± 0.0060 a 1.0636 ± 0.7917 b 0.372 ± 0.2816 b 0.0384 ± 0.0400 a 4 0.1478 ± 0.0597 b 0.0104 ± 0.0115 a 0.0036 ± 0.0034 c 2.8224 ± 0.1081 c 1.9008 ± 0.3453 c 0.0368 ± 0.0132 a 2.5320 ± 0.4117 c 2.5535 ± 0.3375 c 0.0842 ± 0.0673 a 10 2.1620 ± 0.336 c 0.8526 ± 0.5574 b 0.0045 ± 0.0051 c 2.9535 ± 0.1252 d 2.9999 ± 0.0543 d 2.7043 ± 0.3234 b 2.7725 ± 0.4672 d 3.0476 ± 0.0878 d 1.5364 ± 0.4573 b SGM 1 - 0.0048 ± 0.0064 a 0.0017 ± 0.0093 a 0.0071 ± 0.0108 a 0.0028 ± 0.0175 a 0.0013 ± 0.0090 a 0.0143 ± 0.0140 a 0.0200 ± 0.0198 a 0.0080 ± 0.0109 a 0.0243 ± 0.0160 a 2 - 0.0035 ± 0.0081 a 0.0013 ± 0.0082 a 0.0069 ± 0.0105 a 0.0451 ± 0.0287 a 0.0183 ± 0.0120 a 0.0388 ± 0.0298 a 0.2812 ± 0.2362 b 0.1553 ± 0.1297 b 0.2409 ± 0.2440 b 4 0.0349 ± 0.0186 b 0.0070 ± 0.0075 a 0.0124 ± 0.0092 b 0.5936 ± 0.3639 b 0.1290 ± 0.1043 b 0.0304 ± 0.0104 a 0.9074 ± 0.5209 c 0.7608 ± 0.5171 c 0.1571 ± 0.1582 c 10 0.7549 ± 0.3014 c 0.2533 ± 0.1680 b 0.0105 ± 0.0084 b 1.0018 ± 0.4218 c 0.5128 ± 0.4123 c 0.2198 ± 0.0264 b 1.3746 ± 0.6066 d 1.1593 ± 0.5971 d 0.5938 ± 0.4964 d WGJ 1 0.0022 ± 0.0065 a 0.0023 ± 0.0058 a 0.0064 ± 0.0057 a 0.0491 ± 0.0150 a 0.0118 ± 0.0076 a 0.0088 ± 0.0090 a 0.0803 ± 0.0365 a 0.0687 ± 0.0362 a 0.0109 ± 0.0073 a 2 0.0191 ± 0.0110 b 0.0077 ± 0.0081 a 0.0054 ± 0.0053 a 0.1948 ± 0.0485 b 0.0933 ± 0.0313 b 0.0151 ± 0.0056 a 0.1591 ± 0.0710 b 0.3237 ± 0.1053 b 0.0511 ± 0.0251 b 4 0.1242 ± 0.0279 c 0.0573 ± 0.0158 b 0.0065 ± 0.0067 a 0.3319 ± 0.0885 c 0.2099 ± 0.0661 c 0.0730 ± 0.0259 b 0.2182 ± 0.1050 c 0.5222 ± 0.1616 c 0.2142 ± 0.0934 c 10 0.3095 ± 0.0628 d 0.1928 ± 0.0404 c 0.0265 ± 0.0089 b 0.4019 ± 0.1273 d 0.2714 ± 0.1036 d 0.2189 ± 0.1195 c 0.3469 ± 0.1842 d 0.6301 ± 0.1572 d 0.3845 ± 0.1406 d a,b,c,d In columns, values with the same superscript within each culture medium are not significantly different (p > 0.05). Abbreviations: T, temperature in ºC; aw , water activity; YESB, Yeast extract sucrose broth; SGM, Synthetic grape juice medium; WGJ, White grape juice. 26 Table 4. Mean OTA concentration values (in µg/ml) produced by Aspergillus carbonarius strains at each condition assayed and incubation time. OTA (µg / ml) mean value ± (no. of OTA positive strains) culture days T 15ºC 25ºC 35ºC media aw 0.99-0.98 0.95 0.90 0.99-0.98 0.95 0.90 0.99-0.98 0.95 0.90 CYB 1 ND a ND a ND a ND a ND a ND a 0.052 ± 0 a (1) ND a ND a 2 ND a ND a ND a 0.1566 ± 0.2189 b (16) ND a ND a 0.1965 ± 0.2893 a,b (8) ND a ND a 4 ND a ND a ND a 0.9742 ± 1.0972 c (16) 0.3022 ± 0.5395 a,b (12) ND a 0.2828 ± 0.5487 c (15) 0.1145 ± 0.0439 a (2) ND a 10 8.4784 ± 7.361 b (16) 0.6995 ± 1.30 b (14) ND a 0.9958 ± 1.0135 c (16) 1.0181 ± 1.6597 b (10) 0.3883 ± 0.572 b (7) 0.2039 ± 0.2894 b,c (16) 0.1083 ± 0.1607 b (15) 0.069 ± 0.011 a (1) SGM 1 ND a ND a ND a ND a ND a ND a ND a ND a ND a 2 ND a ND a ND a ND a ND a ND a 0.0762 ± 0.0532 b,c (7) ND a ND a 4 ND a ND a ND a 0.1783 ± 0.1459 b (16) 0.0907 ± 0.069 a,b (7) ND a 0.0731 ± 0.0398 b,c (10) 0.061 ± 0.0057 a,b (3) ND a 10 0.1329 ± 0.0902 b (16) 0.0865 ± 0.0934 b (13) ND a 0.1756 ± 0.2119 b (16) 0.122 ± 0.1276 b (13) 0.0694 ± 0.0304 a (4) 0.0775 ± 0.0285 c (15) 0.0613 ± 0.0126 b (6) 0.0586 ± 0.008 a (3) WGJ 1 ND a ND a ND a ND a ND a ND a ND a ND a ND a 2 ND a ND a ND a 0.0919 ± 0.0334 b (16) ND a ND a 0.0724 ± 0.0315 b (15) 0.0596 ± 0.0069 a (4) ND a 4 ND a ND a ND a 0.1175 ± 0.0489 c (16) 0.0626 ± 0.0159 b(11) ND a 0.0727 ± 0.027 b (15) 0.0696 ± 0.0123 a (3) ND a 10 0.098 ± 0.0416 b (16) 0.0799 ± 0.0255 b (16) ND a 0.0884 ± 0.0334 b (16) 0.058 ± 0.0048 b (8) 0.0576 ± 0.0037 a (2) 0.0735 ± 0.0343 b (16) 0.0613 ± 0.0117 b (15) 0.0546 ± 0.0008 b (4) a,b,c In columns, values with the same superscript within each culture medium are not significantly different (p > 0.05). ND, denotes not detected. Abbreviations: T, temperature in ºC; aw , water activity; YESB, Yeast extract sucrose broth; SGM, Synthetic grape juice medium; WGJ, White grape juice. 27 Fig.1 A1995 A3615 A1021 A884 A1653 A1625 A2128 A1841 100 A1002 A1004 A882 A. carbonarius CBS 556.65T (AJ964873) A2275 A2520 A3905 100 A1749 A1687 A. sclerotioniger CBS 115572T (EU163271) 99 A. ibericus CBS 121593 T (AJ971805) A. sclerotiicarbonarius CBS 121057 T (EU159235) A. brasiliensis CBS 101740 T (AM295175) 91 A. niger CBS 554.65T (JX500080) 99 A. lacticoffeatus CBS 101883T (EU163270) 98 A. welwitschiae CBS 139.54T (KC480196) A. tubingensis CBS 134.48T (AJ964876) A. luchuensis CBS 205.80T (JX500071) 98 A. piperis CBS 112811T (EU163267) 81 A. neoniger CBS 115656T (FJ491700) A. eucalypticola CBS 122712 T (EU482433) A. costaricaensis CBS 115574 T (EU163268) A. vadensis CBS 113365 T (EU163269) A. ellipticus CBS 482.65 T (AM117809) 100 A. heteromorphus CBS 117.55 T (AM421461) 70 A. homomorphus CBS 101889 T (AM887865) A. labruscus IBT 33586 T (KT986008) A. saccharolyticus CBS 127449 T (HM853554) A. indologenus CBS 114.60 T (AM419750) 100 A. japonicus CBS 114.51 T (AJ964875) A. uvarum CBS 121591 T (AM745755) 100 72 A. trinidadensis NRRL 62480 (HE984425) A. aculeatinus CBS 121060 T (EU159241) 89 A. aculeatus CBS 172.66 T (AJ964877) 70 A. floridensis IHEM 21069 (HE818083) 73 A. fijensis CBS 313.89 T (FJ491695) 93 A. brunneoviolaceus CBS 621.78 T (EF661147) 0.05 504 505 28 506 Fig. 2 507 A-884 (in CYB) A-1002 (in CYB) 5 20 4 OTA (in ug/ml) 15 OTA (in ug/ml) 3 10 2 5 1 0 0 days 1 2 4 10 1 2 410 1 2 4 10 1 2 4 10 1 2 4 10 1 2 4 10 1 2 4 10 1 2 410 1 2 4 10 days 1 2 4 10 1 2 410 1 2 4 10 1 2 4 10 1 2 4 10 1 2 4 10 1 2 4 10 1 2 410 1 2 4 10 T (ºC) T (ºC) 15 25 35 15 25 35 15 25 35 15 25 35 15 25 35 15 25 35 wa wa 90 95 99 90 95 99 0. 0. 0. (a) 0. 0. 0. (a) 508 A-2128 (in CYB) Monthly maximum and minimum average temperatures in Mediterranean areas and Thailand 2,5 1 2 3 4 5 6 7 8 9 10 11 12 T max (ºC); Mediterranean T max (ºC); Thailand 40 2,0 30 OTA (in ug/ml) 1,5 20 Temperature (ºC) 10 1,0 0 T min (ºC); Mediterranean T min (ºC); Thailand 40 0,5 30 20 0,0 days 1 2 4 10 1 2 410 1 2 4 10 1 2 4 10 1 2 4 10 1 2 4 10 1 2 4 10 1 2 410 1 2 4 10 10 T (ºC) 0 15 25 35 15 25 35 15 25 35 1 2 3 4 5 6 7 8 9 10 11 12 wa 90 95 99 month 0. 0. 0. (a) data obtained from https://weather-and-climate.com (b) 509 510 29