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International Journal of Food Micro

Effect of carbendazim and physicochemical factors on the growth andochratoxin A production of Aspergillus carbonarius isolated from grapes

Ángel Medina a, Rufino Mateo b, Francisco M. Valle-Algarra b,Eva M. Mateo a, Misericordia Jiménez a,⁎

a Dpto. de Microbiologia i Ecologia, Universitat de València, Dr. Moliner 50, E-46100, Burjassot, Valencia, Spainb Dpt. de Química Analítica, Universitat de Valencia, Dr. Moliner 50, E-46100, Burjassot, Valencia, Spain

Received 25 January 2007; received in revised form 13 July 2007; accepted 28 July 2007

Abstract

Carbendazim is a systemic fungicide that is commonly used on several crops (tobacco, fruit, vegetables, cereals, etc.). This fungicide is used tocontrol fungal infections in vineyards. It is indicated against Botrytis cinerea, Uncinula necator, Plasmopara viticola and other fungi and can beused either alone or coupled with other fungicides. However, there is a lack of in-depth studies to evaluate its effectiveness against growth ofAspergillus carbonarius isolated from grapes and OTA production. A medium based on red grape juice was used in this study. Preliminarystudies were performed at 0.98 aw and 25 °C using carbendazim concentrations over a wide range (1–2000 ng/ml medium) to control both growthof a strain of A. carbonarius isolated from grape and its ability to produce OTA. As the lag phase increased considerably at levels N 1000 ng/ml ofmedium, detailed studies were carried out in the range 50–450 ng/ml of medium at 0.98–0.94 aw and 20–28 °C. Statistical analysis (multifactorANOVA) of the data revealed that the three factors assayed and the interactions aw–carbendazim concentration and aw–temperature hadsignificant effects on lag phase duration. The highest lag-times were observed at 0.94 aw, 20 °C, and with 450 ng carbendazim/ml. The threefactors also had significant effects of the growth rate and there was an interaction between aw and temperature. The growth rate of A. carbonariusin these cultures is favoured by high water availability and relatively high temperatures. However, addition of carbendazim at the assayed levelsdid not significantly influenced fungal growth rate. Accumulation of OTA was studied as a function of four factors (the three previouslyconsidered, and time). All factors had significant effects on the accumulation of OTA. There were also two significant interactions (aw–temperature and temperature–time). On the basis of the results obtained, carbendazim does not increase the lag phase of A. carbonarius except atrelatively low aw and temperatures. It does not substantially decrease fungal growth rate once growth is apparent but it appears to cause anincrease in OTA accumulation in the medium at the doses assayed. Carbendazim, which is widely used against fungal infections in grape, canpositively influence OTA production by A. carbonarius in field, which can increase OTA content in grape juices and wines.© 2007 Elsevier B.V. All rights reserved.

Keywords: Aspergillus carbonarius; Carbendazim; Ochratoxin A; Mycotoxins; Fungal growth

1. Introduction

Economic losses arising from plant diseases caused byphytopathogenic fungi are principally associated with yieldreductions. However, fruit, vegetable, and cereal quality andsafety may also be adversely affected, undermining bothconsumer confidence and profitability to the producer. Themain objective for producers and researchers is to avoid the

⁎ Corresponding author. Tel.: +34 963543144; fax: +34 963543202.E-mail address: misericordia.jimenez@uv.es (M. Jiménez).

0168-1605/$ - see front matter © 2007 Elsevier B.V. All rights reserved.doi:10.1016/j.ijfoodmicro.2007.07.053

extended contamination of plant-derived foods and animal feedwith the secondary metabolites of phytopathogenic fungi,particularly mycotoxins.

Mycotoxins are a diverse group of compounds with toxicproperties towards humans and other animals, causing a widerange of acute and chronic effects collectively known as myco-toxicoses and are produced by a wide range of fungi. Many plantpathogenic species of Fusarium, Aspergillus, Alternaria and Pe-nicillium produce mycotoxins of concern in human and animalhealth (D'Mello and Macdonald, 1997; Smith, 1997; Panigrahi,1997; Abramson, 1997). Ochratoxin A (OTA) has been reported

231Á. Medina et al. / International Journal of Food Microbiology 119 (2007) 230–235

to be a nephrotoxic and carcinogenic mycotoxin, produced byvarious species of Aspergillus and Penicillium, such as A.ochraceus and P. verrucosum (Schlatter et al., 1996; Petzingerand Ziegler, 2000). Recently, A. carbonarius has been demon-strated to be the mould responsible for contamination of wine,grapes, grape juice and vine fruits with OTA (Magan and Aldred,2005).

Carbendazim (methyl-2-benzimidazol carbamate) is a system-ic benzimidazole fungicide that plays a very important role inplant disease control. It was first reported in 1973 (Hicks, 1998)and was developed by BASF, Hoeschst and Dupont. It is ametabolite of other fungicides, such as benomyl and is appliedworld-wide on several crops (tobacco, fruit, vegetables, cereals,etc.) to control fungi that can lead to plant disease. It is also used inpost-harvest food storage, in seed pre-planting treatment, and as afungicide in paint, paper and wood. Carbendazim inhibits thepolymerisation of free tubulin molecules by binding an argininresidue of the β-tubulin subunit and acts by disrupting celldivision through linking to the nuclear spindle, which inhibitsfungal growth (IPCS, 1993). This fungicide has been used in thecontrol of fungal infections in vineyards and is indicated againstBotrytis cinerea, Uncinula necator, Plasmopara viticola andother fungi. It can be used either alone or coupled with otherfungicides that exhibit different mechanisms of action.

Mycotoxin biosynthesis is determined by a wide range offactors, broadly classified into physical, biological and chemical,and by interactions involving these factors. Environmentaltemperature, time and humidity are primary factors that interactto affect mycotoxin biosynthesis. When pesticides are used toprotect crops, the implications for mycotoxin production need tobe considered. It has been reported that some fungicides influencepositively or negatively the production of mycotoxins (Boyacio-glu et al., 1992; Gareis and Ceynowa, 1994; Battilani et al., 2003;Bellí et al., 2006).

The aim of this study was to examine the efficacy ofcarbendazim against (a) mycelial extension and (b) OTAaccumulation over time by A. carbonarius on a freshly madegrape juice-based medium at different temperatures (20, 25 and28 °C), carbendazim concentrations (0, 50, 250, 350 and 450 ng/ml) and water activities (0.98, 0.96 and 0.94 aw).

2. Materials and methods

2.1. Fungal isolate

A strain of A. carbonarius isolated from wine grapes andcapable of producing OTA was selected for the study. Thisisolate is deposited in the fungal collection of the Department ofMicrobiology and Ecology at the University of Valencia withreference Ac25.

2.2. Formulation of carbendazim

The carbendazim formulation used in this study was Carzim®(50% w/v a.i.). It was diluted in water and a stock emulsioncontaining 100 mg carbendazim/l was made. This emulsion wasdiluted with water and added to the medium to screen a range of

concentrations from 1 to 2000 ng/ml of medium. Preliminaryexperiments showed that growth of the A. carbonarius isolatewas not satisfactory at higher concentrations (N 1000 ng/ml).Production of secondary metabolites, such as the mycotoxins,may be stimulated if certain environmental stress factors and lowfungicide doses are maintained during growth of mycotoxin-producing fungi (Placinta et al., 1996; D'Mello et al., 1998;Magan et al., 2002). Therefore, detailed studies were subse-quently carried out over a narrower range of carbendazimconcentration.

2.3. Growth studies

A freshly stock extract from red wine grapes was prepared. Itwas divided into three portions and each portion was modifiedby mixing 20% extract with 80% of a mixture of water andglycerol of variable composition to provide aw values of 0.98,0.96 or 0.94 in the definitive solid media (after addition of 2%w/v of agar). The pH was adjusted at 4.5 by careful addition ofNaOH solution and using a pH-meter. Once the agar was added,the media were autoclaved (115 °C, 30 min). Then, appropriatevolume of carbendazim stock solution was added around 45 °Cto obtain the desired concentration in the medium, which wasvigorously shaken and poured into Petri dishes. The respectiveaw values were checked after sterilisation using uninoculatedPetri dishes. Water activities were measured using a NovasinaRTD 502 apparatus (Novasina GmbH, Pfäffikon, Switzerland).The strain of A. carbonarius was grown on Potato DextroseAgar for 7 days at 28 °C. Then, a suspension of 1·106 spores/mlwas prepared in 0.9% NaCl solution and used to inoculate thecentre of the Petri dishes under sterile conditions. Theincubation temperatures were 20, 25 and 28 °C, based on theinformation indicating that optimum temperature for OTAproduction is approximately 20 °C whereas that for growth isnear 30 °C (Mitchell et al., 2004). Incubation was performed inclosed chambers where there were beakers containing glycerol–water solutions of the same aw, according to Llorens et al.(2004). Mycelia extension rates were measured over time untilcolonies occupied all the plate. Lag phase for growth wasconsidered as the time (days) to reach a colony 5 mm diameter.For each treatment, ten plates were prepared and two right-angled diameters of the colonies were randomly chosen andmeasured every day until the colony filled the whole Petri dishor the cultures were used to analyse OTA. The sum of twodiameters was divided by 4 to provide the radius. These radiusmeasurements were then averaged over the number of measureddishes. Linear regression of colony radius (mm) against time(days elapsed from the day the colony was 5 mm diameter) wasused to determine growth rates (mm/day).

2.4. Ochratoxin A analysis

Twenty grams of each fungal culture (agar+ fungal biomass)was cut into small pieces and extracted with 50 ml methanol(Sigma–Aldrich, Alcobendas, Spain) by shaking at 110 rpm for1 h in orbital shaker at 25 °C in the dark. The extracts werefiltered through filter paper (Whatman No. 4) containing 5–10 g

Table 2Effect of different concentrations of carbendazim in a grape juice-based solidmedium (pH 4.5) modified with glycerol to 0.94, 0.96 and 0.98 aw on thegrowth rate (mm/day) of a strain of Aspergillus carbonarius

Carbendazim(ng/ml)

aw

0.94 0.96 0.98

Temperature (°C) Temperature (°C) Temperature (°C)

20 25 28 20 25 28 20 25 28

0 5.13 7.80 11.53 6.34 11.54 14.46 5.64 10.27 13.5250 5.34 10.29 11.82 6.40 11.38 14.20 5.31 10.21 13.75250 5.09 10.37 10.07 6.41 10.77 13.55 5.07 10.23 13.87350 5.29 9.35 9.57 6.28 9.67 14.00 4.04 10.86 13.73450 5.20 9.28 7.99 5.59 9.03 12.55 3.51 9.01 13.22

232 Á. Medina et al. / International Journal of Food Microbiology 119 (2007) 230–235

of Celite 545 (Sigma–Aldrich). One milliliter of each filteredextract was removed and centrifuged at 1100 rpm for 15 min forfurther purification. The supernatant of each tube was removedand placed in an amber vial for LC analysis.

Once fungal growth was detected (colony diameter N 5 mm),level of OTA in the cultures was determined at 3, 7, and 15 days.Therefore, the actual day for OTA accumulation was lag-time(days)+day of OTA analysis. This protocol was performed eachtime with two dishes from the same treatment.

The LC system was a Waters 600E system controller, a Mil-lipore Waters 717 plus autosampler and a Waters 470 scanningfluorescence detector (Waters, Milford, MA, USA). Excitationand emission wavelengths were 330 and 460 nm, respectively.The samples were separated using a C18 Phenomenex Gemini ®column (150×4.6 mm, 5 μm particle size) (Phenomenex,Macclesfield, UK), with a guard column of the same material.Run time for samples was 20 min with OTA being detected atabout 12 min. The flow rate of the mobile phase (acetonitrile–water–acetic acid; 44:55:1, v/v/v) was 1 ml/min. Standards usedwere in the 10–1000 ng/ml range. The recovery rate for OTAwas89% from the agar-based medium with a limit of detection ofb 0.01 μg OTA/g medium, based on a signal-to-noise ratio of 3:1.Measurementswere processed using a computerwithMillennium® 4.0 software (Waters).

2.5. Statistical analysis

Statistical analysis was performed using Statgraphics 5.1Plus Professional Edition (Statpoint Inc., Herndon, VA, USA).

3. Results and discussion

3.1. Lag phase

The lag-times observed from the experiment are shown inTable 1. The effect of aw, temperature and the concentration ofcarbendazim can be seen on the time elapsed betweeninoculation and the time when fungal growth became evident.

The statistical analysis of the data (ANOVA) revealed that thethree factors assayed and aw–carbendazim concentration andaw–temperature interactions had significant effects on lag phaseduration (p-values b 0.05). Using Tukey–honestly significantdifference (Tukey–HSD) multiple range test at 95% confidence

Table 1Effect of different concentrations of carbendazim in a grape juice-based solidmedium (pH 4.5) modified with glycerol to 0.98, 0.96 and 0.94 aw on the lag-time (days) of Aspergillus carbonarius

Carbendazim(ng/ml)

aw

0.94 0.96 0.98

Temperature (°C) Temperature (°C) Temperature (°C)

20 25 28 20 25 28 20 25 28

0 3 2 2 3 2 2 1 1 150 3 2 2 3 2 2 1 1 1250 4 3 2 4 3 2 1 1 1350 7 3 3 4 3 3 1 1 1450 7 4 3 5 3 3 1 1 1

level, the cases were grouped into homogeneous groups withregard to the different parameters used. The aw level providedtwo different clusters (0.98 aw and 0.94–0.96 aw). Mould lagphase was only one day at the highest aw-value so visiblemycelium development was obvious in all cultures regardless ofthe remaining factor levels the day following incubation.According to the Tukey–HSD treatment, the lag phases obtainedat 20 °C were included in one group whereas those obtained at25–28 °C were included in another homogeneous group. Thus,the lowest temperature assayed (20 °C) produced a significantincrease in lag phase with respect to 25–28 °C, these lattertemperatures showing no significant influence on lag phaseduration. The concentration of carbendazim split the cases intotwo homogeneous but overlapping groups, one formed by 0(control), 50, 150, 250, and 350 ng/ml (ppb) and the other by250, 350 and 450 ng/ml. The highest level (450 ng/ml) produceda significant increase in fungal lag phase compared with blanksor 50 ng/ml. The carbendazim level influenced the duration ofthis phase, which was 1–3 days for blanks and 1–7 days for450 ng/ml cultures.

Therefore, the three studied factors influenced the lag phaseduration, which was reduced by high water availability andrelatively high temperatures (near 28 °C) while carbendazimlevels in the interval 50–250 ng/ml did not show significanteffects. The highest lag-times were observed at 0.94 aw, 20 °C,and with 450 ng carbendazim/ml. The fungicide effect,however, is not strong at the assayed level.

Mitchell et al. (2004) studied the growth evolution of eightstrains of A. carbonarius from various countries and found thatlag-times increased with a reduction of aw and temperature; infact, when they used 10 °C and 0.85 aw, lag-times exceeded20 days. Our results agree with these findings although the growthconditions used in our experiments were not so restrictive.

3.2. Growth rate

The growth rates obtained in the different experimentsare shown in Table 2. Multifactor ANOVA shows that the threefactors had significant effects on fungal growth rate and thatthere was a significant interaction between aw and temperature(p-values b 0.05); however, p-value for carbendazim was0.0413, which indicates that this factor was the least influential.

Fig. 1. Evolution with time (elapsed days after lag phase) of the OTA level (ng/g orppb) accumulated in the grape-based solid culture medium at 0.98 aw, at threetemperatures (28, 25 and 20 °C) and different carbendazim levels.

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The Tukey–HSD multiple range test (P=95%) led to threehomogeneous non-overlapping groups with respect to temper-ature. Thus, keeping aw and fungicide concentration constant,the higher the temperature tested (within 20–28 °C), the higherthe observed growth rate.

The Tukey–HDS test (P=95%) displayed two homogeneousgroups with regard to aw; one of them was formed by the two

Table 3OTA accumulation (ng/g) in the culture medium on the day 15 from the lag-time

Carbendazim(ng/ml)

aw

0.94 0.96

Temperature (°C) Temperat

20 25 28 20

0 324.5 122.7 31.4 102850 273.8 81.2 28.4 1211250 235.3 171.1 38.6 1365350 542.3 121.8 123.9 1476450 161.6 366.1 661.9 1928

highest values and the other by 0.94 aw. The latter value produceda significant decrease in the growth rate of A. carbonarius.

The same test applied to the carbendazim level in the mediumgave rise to two homogeneous groups although there was aconsiderable degree of overlapping between them (0, 50, 250 and350 ng/ml) and (0, 250, 350 and 450 ng/ml). Carbendazim at50 ng/ml level had no negative effect on the growth rate of A.carbonarius as compared with the blank. Except between 50and 450 ng/ml, there were no significant differences among thecarbendazim levels on growth rate.

With the aim of predicting fungal growth rate as a function ofthe assayed factors, a possible model was computed by multipleregression analysis. Growth rates were transformed into theirnatural logarithms [Log (growth rate)] to obtain the best fit. Theequation that provided the highest value of r2 was:

Log ðgrowth rateÞ ¼ �329:437 � 0:78267⁎ðtÞþ 70644⁎ðawÞ� 7:6804E

� 7⁎½Carbendazim�2

� 0:006699⁎ðtÞ2

� 382:789⁎ðawÞ2þ 1:26023⁎ðtÞ⁎ðawÞ ð1Þ

Where t is temperature. The coefficients of each term indicatethe relevance of the different factors. The last term accounts forthe interaction between t and aw.

The r2 statistic (0.9552) indicates that the model as fittedexplains 95.52% of the variability in Log(growth rate).

It can be seen in Table 2 that the growth rate ofA. carbonariusin these cultures is favoured by high water availability andrelatively high temperatures. However, addition of carbendazimat the levels assayed had no significant influence on fungalgrowth rate.

3.3. OTA accumulation

Accumulation of OTA was studied as a function of fourfactors (aw, carbendazim concentration, temperature and time).Fig. 1 shows OTA concentration in the culture medium atdifferent days, temperatures and carbendazim levels maintainingaw=0.98 and Table 3 lists the OTA levels in the culture mediumon the day 15 from the lag-time. The statistical treatment of the

0.98

ure (°C) Temperature (°C)

25 28 20 25 28

326.7 126.7 2743 2006 862.4283.2 79.8 3437 2022 815.6524.5 123.2 4449 2163 972.9634.2 158.9 5122 2757 1696885.9 413.5 5955 4301 2058

Fig. 2. Plot of OTA levels observed by LC analysis against the values predictedby the equation obtained by multiple linear regression.

234 Á. Medina et al. / International Journal of Food Microbiology 119 (2007) 230–235

data by multifactor ANOVA showed that all factors had sig-nificant effects on OTA accumulation (p-values b 0.005). Therewere also two significant interactions (aw–temperature andtemperature–days).

The Tukey–HSD test at the 95% confidence level producedseveral groupings. The aw level led to three homogeneous non-overlapping groups. The highest levels of OTA were found at0.98 aw and decreased when aw decreased. Thus, the amount ofavailable water positively influences the accumulation (andproduction) of OTA.With regard to the time, three homogeneousnon-overlapping groups (days 3, 7 and 15) were noticeable. OTAaccumulates in the medium with time continuously until day 15.With respect to temperature two homogeneous groups appeared;one of them formed by 25 and 28 °C, and another by 20 °C, whichwasmore favourable for OTAproduction. A double groupingwasalso detected by the Tukey–HDS test (P=95%) with regard tocarbendazim level. The two groups overlapped as in the case offungal growth rate. One group corresponded to cultures thatcontained 0–350 ng carbendazim/ml and the other to cultureswhere carbendazim concentrations were 250–450 ng/ml. Theconcentration of this fungicide positively influenced OTAaccumulation so that the higher the carbendazim level, the higherthe OTA accumulation in the medium.

A possible model for OTA accumulation as a function of thestudied parameters was computed by multiple regressionanalysis. The best equation was:

½OTA� ¼ 602931:0 � 1:44084E6⁎ðawÞ� 52:6544⁎½carbendazim�þ 688:671⁎ðdaysÞ þ 5739:18⁎ðtÞ� 5888:28⁎ðawÞ⁎ðtÞ � 15:6813⁎ðtÞ⁎ðdaysÞþ 55:9924⁎½carbendazim�⁎ðawÞþ 842763:0⁎ðawÞ2 � 10:1374⁎ðdaysÞ2 ð2Þ

Where t is the temperature (°C) and days refer to days afterthe lag phase.

The r2 statistic (0.7743) indicates that the model as fittedexplains 77.43% of the variability in OTA accumulation in themedium by our strain.

Fig. 2 shows the plot of OTA levels observed by LC analysisagainst the values predicted obtained by Eq. (2) and the equality(observed=predicted) line. As can be seen, predictability is a

difficult task, especially at low and high OTA levels. Residuals(differences between observed and predicted) are usuallypositive beyond 2000 ng OTA/ml.

Thus, carbendazim effects at the assayed levels (50–450 ng/ml) can be summarized as follows: it does not increase the lagphase of our strain of A. carbonarius except at relatively low awand temperatures, although the largest delay was only 4 days withrespect to control. It does not substantially decrease fungal growthrate once growth is apparent but it appears to increase OTAaccumulation in the medium. The possible mechanism of car-bendazim effect on OTA production is not known. However,reduction of OTA and ochratoxin B production in cultures of A.ochraceus in yeast extract–sucrose medium and in maize kernelshas been reported when dichlorvos (an organophosphatepesticide) was added at levels of up to 300 mg/l broth or/kgcorn (Wu and Ayres, 1974). As carbendazim is widely usedagainst fungal infections in grapevine before grape harvest itmight also positively influence OTA production by A. carbonar-ius in field, which can increase theOTA content in grape juice andwines. Battilani et al. (2003) have found that some fungicides canstimulate OTA synthesis in cultures of OTA producing strainsbelonging to Aspergillus section Nigri.

It has been reported that the fungicides fluazinam and procy-midone have an OTA-enhancing effect inA. carbonarius culturesin synthetic grape-like medium. OTA production was increased,although not significantly, when inorganic fungicides containingsulphur were added to the medium (Bellí et al., 2006). Accordingto Tjamos et al. (2004) carbendazim was not effective controllingthe A. carbonarius population in vineyards so that this specieswas prevailing over A. niger aggregate when this fungicide wasapplied. Therefore, carbendazim cannot be considered a goodfungicide in vineyards because it promotes A. carbonarius pre-dominance over other less ochratoxigenic species and can in-crease OTA production as proved in that paper under in vitroconditions.

With regard to the effect of aw and temperature on the growthof A. carbonarius and OTA production, the results in this paperagree with those from other authors (Marín et al., 2006; Leonget al., 2006), as growth is favoured by high aw (0.98) and tem-peratures while OTA production is increased at mild temperature(approximately 20 °C) and 0.96–0.98 aw.

Acknowledgements

The authors are grateful to the SpanishMinistry for Educationand Science (Project AGL-2004-07549-C05-02/ALI) for thefinancial support. The authors would also like to thank theValencian Regional Government (Conselleria d'Empresa, Uni-versitat i Ciencia) for financing a research grant.

References

Abramson, D., 1997. Toxicants of the genus Penicillium. In: D'Mello, J.P.F.(Ed.), Handbook of Plant and Fungal Toxicants. CRC Press, Boca Raton,Florida, pp. 303–317.

Battilani, P., Pietri, A., Bertuzzi, T., Formenti, S., Barbano, C., Languasco, L.,2003. Effect of fungicides on ochratoxin producing black aspergilli. Journalof Plant Pathology 85, 285–291.

235Á. Medina et al. / International Journal of Food Microbiology 119 (2007) 230–235

Bellí, N., Marín, S., Sanchis, V., Ramos, A.J., 2006. Impact of fungicides onAspergillus carbonarius growth and ochratoxin A production on syntheticgrape-like medium and on grapes. Food Additives and Contaminants 23,1021–1029.

Boyacioglu, D., Hettiarachchy, N.S., Stack, R.W., 1992. Effect of three systemicfungicides on deoxynivalenol (vomitoxin) production by Fusariumgraminearum in wheat. Canadian Journal of Plant Science 72, 93–101.

D'Mello, J.P.F., Macdonald, A.M.C., 1997. Mycotoxins. Animal Feed Scienceand Technology 69, 155–166.

D'Mello, J.P.F., Macdonald, A.M.C., Postel, D., Dijksma, W.T.P., Dujardin, A.,Placinta, C.M., 1998. Pesticide use and mycotoxin production in Fusariumand Aspergillus phytopathogens. European Journal of Plant Pathology 104,741–751.

Gareis, M., Ceynowa, J., 1994. Influence of the fungicide Matador (tebucona-zole/triadimenol) on mycotoxin production by Fusarium culmorum.Zeitschrift für Lebensmittel-Untersuchung und -Forschung 198, 244–248.

Hicks, B., 1998. Generic Pesticides — The Products and Markets. AgrowReports. P.J.B. Publications.

IPCS (International Programme on Chemical Safety), 1993. Health and SafetyGuide No. 82. Carbendazim Health and Safety Guide. United NationsEnvironment Programme International Labour. World Health Organization,Geneva.

Leong, S.L., Hocking, A.D., Scott, E.S., 2006. Effect of temperature and wateractivity on growth and ochratoxin A production by Australian Aspergilluscarbonarius and A. niger isolates on a simulated grape juice medium.International Journal of Food Microbiology 110, 209–216.

Llorens, A., Mateo, R., Hinojo, M.J., Valle-Algarra, F.M., Jiménez, M., 2004.Influence of environmental factors on the biosynthesis of type B trichothecenesby isolates of Fusarium spp. from Spanish crops. International Journal of FoodMicrobiology 94, 43–54.

Magan, N., Aldred, D., 2005. Conditions of formation of ochratoxin A in drying,transport and in different commodities. Food Additives and Contaminants22, S10–S16.

Magan, R., Hope, R., Colleate, A., Baxter, E.S., 2002. Relationship betweengrowth and mycotoxin production by Fusarium species, biocides andenvironment. European Journal of Plant Pathology 108, 685–690.

Marín, S., Bellí, N., Lasram, S., Chebil, S., Ramos, A.J., Ghorbel, A., Sanchis,V., 2006. Kinetics of ochratoxin a production and accumulation by Asper-gillus carbonarius on synthetic grape medium at different temperaturelevels. Journal of Food Science 71, M196–M200.

Mitchell, D., Parra, R., Aldred, D., Magan, N., 2004. Water and temperaturerelations of growth and ochratoxin A production by Aspergillus carbonariusstrains from grapes in Europe and Israel. Journal of Applied Microbiology97, 439–445.

Panigrahi, S., 1997. Alternaria toxins. In: D'Mello, J.P.F. (Ed.), Handbook ofPlant and Fungal Toxicants. CRC Press, Boca Raton, Florida, pp. 319–337.

Placinta, C.M., Macdonald, A.M.C., D'Mello, J.B.F., Harling, R., 1996. Theinfluence of carbendazim on mycotoxin production in Fusarium sporo-trichioides. Proceedings of the Brighton Crop Protection Conference 1996:Pests and Diseases. British Crop Protection Council, Farnham, UnitedKingdom, pp. 415–416.

Petzinger, E., Ziegler, K., 2000. Ochratoxin A from a toxicologicalperspective. Journal of Veterinary Pharmacology and Therapeutics 23,91–98.

Schlatter, C.H., Struder-Rohr, J., Rasonyi, T.H., 1996. Carcinogenicity andkinetic aspects of ochratoxin A. FoodAdditives and Contaminants 13, 43–44(Suppl.).

Smith, J.E., 1997. Aflatoxins. In: D'Mello, J.P.F. (Ed.), Handbook of Plant andFungal Toxicants. CRC Press, Boca Raton, Florida, pp. 269–285.

Tjamos, S.E., Antoniou, P.P., Kazantzidou, A., Antonopoulos, D.F., Papageorgiou,I., Tjamos, E.C., 2004. Aspergillus niger and Aspergillus carbonarius incorinth raisin and wine-producing vineyards in Greece: population composi-tion, ochratoxin A production and chemical control. Journal of Phytopathology152, 250–255.

Wu, M.T., Ayres, J.C., 1974. Effects of dichlorvos on ochratoxin production.Journal of Agricultural and Food Chemistry 22, 536–537.