molecular and chemical monitoring of growth and ochratoxin a biosynthesis ofp. verrucosum in wheat...

Mycotoxin Research Vol. 23, No. 3 (2007), 138-146

Molecular and chemical monitoring of growth and ochratoxin A biosynthesis of P. ve r rucosum in wheat stored at different moisture conditions

M. Schmidt-Heydt 1, W. Richter z, M. Michulec 3, G. Buttinger 3, R. Geisen 1

1 Federal Research Centre for Nutrition and Food, Location Karlsruhe, Haid-und-Neu-Str. 9, 76131 Karlsruhe, Germany

2 Bavarian State Research Centre for Agriculture, Institute of Animal Nutrition and Forage, Prof. D0rrwaechter-Platz 3, 85586 Poing, Germany

3 European Commission, Joint Research Centre, Institute for Reference Materials and Measurements, Retieseweg 111, 2440 Geel, Belgium

Abstract

The growth and ochratoxin A producing activity at the phenotypical and molecular level of P. verrucosum in wheat stored at different relative moisture conditions have been followed. Growth has been measured by determination of colony forming units (cfu). The expression of the ochratoxin A polyketide synthase (otapksPV) has been followed over time by reverse transcriptase Real Time PCR, whereas the biosynthesis of ochratoxin A by the fungus has been followed by HPLC. The remoisted and inoculated wheat was kept in perforated plastic bags and stored in tower silos. The bags were adjusted to 24%, 19% and 14% (m/m) relative moisture and stored for 6 month (September 06 - February 06) at ambient temperatures. A high increase of the cfu number up to a level of about lxl08 cfu/g could be observed in the 24% sample. Interestingly no growth could be detected in the 14% and 19% samples. This result was supported by the quantitative Real Time PCR data. In congruence with this growth behaviour ochratoxin A could be detected from the first to the last time point in the 24% sample. Interestingly the ochratoxin A amounts measured over time did not show increasing or constant values, but varied from higher to lower values. After storage of three month (November 2006) the ochratoxin A concentration in the wheat was highest. The expression of the otapksPV gene mirrors this behaviour. The expression of this gene also fluctuates around a certain value, showing high activity after three month of storage.

Keywords: P. verrucosum, ochratoxin A, relative moisture, polyketide synthase gene expression

Introduction

Ochratoxin A is a fungal mycotoxin composed of the polyketide dihydroisocoumarin and the amino acid phenylalanine. Ochratoxin A is an important mycotoxin with toxic activities mainly against the kidneys (1). It is rated by the WHO as a type B carcinogen. For this reason the EU recently set statutory limits for ochratoxin A in certain food commodities (2). Especially cereals, coffee, cocoa, wine, grape juice, spices or meats are prone to be contam-

Presented at the 29 th Mykotoxin-Workshop, Fellbach, Germany, May 14-16, 2007

Correspondence: Rolf Geisen, Federal Research Centre for Nutrition and Food, Location Karlsruhe, Haid-und-Neu-Str. 9, 76131 Karlsruhe, Germany ([email protected])

Financial support: Part of the work was supported by the EU project: "Development of cost-effective control and prevention strategies for emerging and future food borne pathogenic microorganisms throughout the food chain" (PathogenCombat FOOD-CT-2005-07081 )

Received 2 Aug 2007; accepted 29 Oct 2007

inated by ochratoxin A (3, 4, 5, 6, 7). The most important Aspergillus species able to produce ochratoxin A are A. ochraceus, A. wester- dijkiae, A. steynii occurring in coffee (8, 9) and A. carbonarius and A. niger on grapes (10). In contrast to the Aspergilli, which occur mainly in regions with warmer climate, the Penicillia prefer lower optimum growth temperatures. P. nordicum is a species, which is morphologi- cally very similar to P. verrucosum, but has distinctive differences (11, 12). Both species also occupy different habitats. Whereas P. nordicum is a contaminant of fermented foods with a certain salt content (3, 13), P. verrucosum can be ultimately found on cereals and cereal products (3). According to a recent survey cereal and cereal products contribute most to the intake of ochratoxin A by the consumer (14) indicating the importance of this species for human and animal health. Lindblad et al. (15) demonstrated that a contamination by P. verrucosum indicates the presence of ochra-

138


toxin A in dependence of the water activity. At high water activity levels cfu numbers of P. verrucosum of about 103-104/g indicates the biosynthesis of ochratoxin. At lower cfu levels a higher contamination by P. verrucosum is needed to indicate the biosynthesis of ochratoxin A with the same probability. This result demonstrates the importance of the water content of a sample on growth of P. verrucosum. After drying wheat to 13%-14% moisture content, P. verrucosum is usually not able to grow (16). For this reason moisture contents at or below 14.5% are regarded as safe (17). Ac- 'cording to Gareis and R6del (18) P. verrucosum is not able to produce ochratoxin A below 19%, which is equivalent to a water activity of 0.83. Recently a comprehensive molecular system to analyse the behaviour of P. verrucosum in wheat has been described (19). This system consists of a PCR for identification and detection of P. verrucosum, a Real Time PCR for quantification and a reverse transcriptase Real Time PCR for monitoring gene expression. The whole system is based on the ochratoxin A polyketide synthase gene (otapksPV) of P. verrucosum. In the current work this approach has been used to analyse growth and activation of an ochratoxin biosynthetic gene during different moisture conditions. In parallel ochratoxin A was determined by HPLC.

Materials and Methods

Strains and culture conditions For all experiments P. verrucosum BFE808 from the culture collection of the Federal Research Centre for Nutrition and Food was used. This is a high ochratoxin A producing strain. Strain propagation and maintenance was done on malt extract medium (Merck, Darm- stadt, Germany) with 5 g/1 glucose.

Inoculation o f wheat and storage conditions Six 24 kg lots of wheat (harvest 2006, cultivar Dekan) were inoculated with a spore suspen- sion of P. verrucosum in a way, that the final concentration of spores reached 10 6 spores per gram. The wheat samples were adjusted to the defined moisture contents of 24%, 19% and 14%. The lots were split into two portions and kept into perforated plastic bags which were kept as three duplicates in six steel granary tower silos. Each replicate contained a resis- tance thermometer (Pt 100) and a gas-sampling

tube (20). During the trial the temperature was recorded every four hours. Each month C O 2

concentration was measured and samples were taken as well as analysed for ochratoxin A content, fungal growth, otapksPV expression, and moisture. The experiment was carried out at least 2 times beginning September 2006.

Determination o f the colony forming units (cfu) For determination of the colony forming units 10 g of stored wheat were mixed with 90 ml tween 80 solution (9 g NaC1/1; 1 g Tween 80/1; 1 g agarose/1) and diluted serially tenfold in the same solution. A volume of 100 ~tl was plated out on malt extract agar plates and incubated at 20 ~ for 5 to 7 days. P. verrucosum colonies were easily recognized due to their morpholo- gy and colour under these conditions. They were counted and the cfu levels were calculated.

Isolation o f fungal DNA from wheat samples To isolate fungal DNA directly from wheat samples, an amount of 2 g contaminated wheat was transferred into a mortar, frozen with liquid nitrogen and ground to a powder with the aid of a pestle. All subsequent isolation steps were essentially the same as described earlier (21).

Isolation o f RNA from wheat samples To perform Real Time PCR experiments, RNA has been isolated by using the RNAeasy plant mini kit (Qiagen, Hilden, Germany). An amount of 2 g of the contaminated wheat kernels was ground in a mortar in the presence of liquid nitrogen. About 250mg of the resulting wheat powder were used for isolation of total RNA. The powder was resuspended in 750 ~tl lysis buffer, mixed with 7.5 ~tl [3- mercaptoethanol and about 100 glass beads with a diameter of 1 mm (B. Braun Biotech International GmbH, Melsungen, Germany) in a 2 ml RNase free micro reaction tube. The probe was mixed thoroughly and incubated for 15rain at 55 ~ and 42kHz in an S10H ultrasonic bath (Elma, Singen, Germany). All further procedures were essentially the same as suggested by the manufacturer of the kit.

Real Time PCR to quantify P. verrucosum in wheat The Real Time PCR reactions were performed in a GeneAmp 5700 | Sequence Detection Sys- tem (PE Applied Biosystems, Foster City,

139


USA). The SybrGreen system with two primers was used. The optimal primers and the internal probe used in the reaction were identi- fied within the sequenced 732 bp otapksPV fragment by the Primer Express 1.0 software (PE Applied Biosystems). The primer set had the following nucleotide sequences: otapksPV 1-SYBRI-for, 5'-TTGCGAATCAG GGTCCAAGTA-3'; otapksPV 1-SYBR 1-rev, 5'-CGAGCATCGAAAGCAAAAACA-3': For the PCR reaction the qPCR core kit for SYBR Green (Eurogentec, Liege, Belgium) was used according to the recommendations of the manufacturer. For each reaction 1 gl of the DNA sample solution was mixed with 2.5 gl reaction buffer, 1.5 gl MgCI2 (50 mM); 1.0 gl dNTP-Mix (2.5 raM), 1 gl of each primer (20 ~tM), SYBR green 0.75 gl, Amp Erase LING 0.25 I.tl; Ampli Tag Gold 1.0 ~1, aqua bidest 15.88 gl. The following temperature profile was used: lx (50 ~ 2 min); lx (95 ~ 10 rain); 35x 95 ~ 20 s, 55 ~ 20 s, 72 ~ 30 s); lx (55 ~ 20 s); lx (72 ~ 30 s). To generate the standard curve, the cloned 732 bp otapksPV fragment as larger PCR fragment was amplified by PCR. The concentration of this standard PCR product was determined in a fluorometer (DyNa Quant 200, Pharmacia, Uppsala, Sweden) and the number of copies was calculated. These stock solutions were diluted serially by a factor of 10 and an aliquot of the dilutions was used as a copy number standard during each setup of the Real Time PCR reaction. The concentration of unknown samples was calculated by the GeneAmp 5700 | system according the generated standard c u r v e .

cDNA synthesis For cDNA synthesis 1 gg of the DNase I treat- ed total RNA was used along with the Omni- script Reverse Transcription kit (Qiagen, Hilden, Germany). The reaction mixture was composed essentially as described by the manufacturer and incubated at 37 ~ for 1 h. The cDNA was either directly used for Real Time PCR or stored at -20 ~

Reverse transcriptase Real Time PCR The Real Time PCR reactions to measure the expression of the otapksPV-gene in P. verrucosum contaminated wheat samples were performed essentially as described in Geisen et al. (22).

Quantification of ochratoxin A in wheat by HPLC An in house validated method based on EN 14132 (23) was used to quantify the ochratoxin A in the wheat samples. The samples were ground with a Retsch ZM200 mill (Retsch, Hahn, Germany) through a 0.5 mm sieve. A 20 g aliquot was taken and extracted with 50 ml acetonitrile/water (60+40, v+v) for 60 min. The extract was filtered through a fluted filter and 5 ml of it diluted with 45 ml PBS buffer. For the higher contaminated 24% samples 2.5 ml were diluted with 197.5ml PBS buffer. An amount of 20 ml of the diluted extract was loaded onto an immunoaffinity column (VI- CAM, Watertown, USA) for clean up. The columns were rinsed with 10 ml PBS buffer followed by 10 ml of bidistilled water. Ochra- toxin A was eluted with 1.5 ml methanol/acetic acid (98+2, v+v). The eluate was brought to dryness and redissolved in 0.4 ml acetonitrile/water/acetic acid (399+599+2, v+v+v). Chromatographic separation was performed isocratic on an Alltima HP C18 100x2.1 mm, 3 g m (Alltech, Deinze, Belgium) column with acetonitrile/water/acetic acid (399+599+2, v+v+v) at a flow rate of 0.5 ml/min employing a Dionex GP40 pump (Dionex, Amsterdam, The Netherlands). Ochratoxin A was detected with a fluorescence detector Jasco FP-920 (Jasco, ljsselstein, The Netherlands) with Xex = 330 nm and Xem=460 nm. The methods characteristics are a limit of detection of 0.09 gg/kg, a limit of quantification of 0.27 ~g/kg, a recovery rate of 97% and an expanded relative uncertainty of 8.8% (k=2). The results were normalized to a water content of 11% (m/m) (measured using Karl-Fischer titration). The water content was determined in the samples after milling, due to significant changes of the water content during the milling process.

Results

Monitoring the growth of P. verrucosum in wheat stored with different moisture conditions by determining the cfu P. verrucosum was inoculated to the same initial spore content in all wheat samples (10 6 spores/g), which were then stored for 6 months at different moisture conditions. The growth of P. verrucosum was followed by determining

140


Figure 1. Development of the cfu values of P. verrucosum during storage of wheat with different moisture contents

changes in cfu each month. The results are shown in Figure 1. As expected essentially no growth could be observed in the samples stored with 14% moisture content. The cfu values even reduced to 103-104 per gram and stayed constant for the whole observation period. The same was true for the samples stored with a moisture content of 19%. In contrast the samples stored at 24% moisture content showed immediately an increase in cfu. Already at the first time point (after 1 month) the cfu reached a level of 107 cells/g. This level is further increased up to lxl08 during the following 6 month storage time, indicating active growth and metabolism of P. verrucosum under these conditions.

Monitoring the growth of P. verrucosum in wheat stored with different moisture conditions by molecular methods A previously described PCR approach (19) was used to analyse the wheat samples stored at different moisture conditions to check its applicability to detect the presence of P. verrucosum in wheat. The results are shown in Figure 2. Only in case of the 24% samples the PCR gave positive results confirming the high contamination of these samples by P. verrucosum. By using quantitative Real Time PCR positive reactions could be observed in samples with cfu values above 103-104 (Table 1), e.g. with the samples stored at 24% moisture content, but not with the 14% and

Figure 2. PCR detection of P. verrucosum by con- ventional PCR. Only the samples stored at 24% moisture content showed a clear PCR signal (lanes 7, 8, 13, 15). The lane 16 showed the control

Table 1. Comparison of Real Time PCR results with cfu values of wheat samples stored at different moisture contents

Wheat cfu/g otapksPV otapksPV sample measured 1 calculated 2

14% 2x103

19% 3x103

24% 1.7x10 ~ 4617 4.6x108

~copy number of the otapksPV gene determined in one Real Time PCR reaction 2calculated copy number of the otapksPV gene in one gram of stored wheat

141


19% samples. The observed Real Time PCR values, e.g. the copy numbers of the otapksPV gene, are tenfold higher compared to the cfu values.

Expression of the otapksPV gene and ochratoxin A biosynthesis during storage of wheat In agreement with the growth data no ochratoxin A could be detected in the wheat samples stored at 14% and 19% moisture content (Table 2). However already at the first time point (1 month) high ochratoxin A levels could be detected in the 24% samples (Figure 3A). In fact obviously already at this time point sat- urated levels of ochratoxin A are produced. During the subsequent months of storage no clear increase in the ochratoxin A concentration in this samples could be observed. The ochratoxin concentration rather fluctuates around a certain average value. Interestingly after 3 month of storage (November 06) the highest concentration of ochratoxin A could be found. This is in complete correspondence to the expression of the otapksPV gene (Figure 3B). The transcription levels of that gene are low, but it is constantly expressed over the whole observation period. Also in this case the levels fluctuate around a certain average value, instead having a clear tendency. After 3 month

of storage (November 06) also the highest expression value could be observed, which could explain the high biosynthesis of ochratoxin A at or before that time point. The time course of the temperature during storage is shown in Table 3. Interestingly the temperature drops from around 20 ~ in October to 11 ~ in November, indicating a possible correlation between this shift in temperature and the activation of the otapksPV gene. It is remarkable that during the whole time course a reduction of the ochratoxin A content could be observed for several times. This indicates some kind of degradation of the ochratoxin A produced during storage, either by the fungus or by external activities.

Influence of temperature on the expression of the otapksPV gene Because of the possible effect of low temperatures on the activation of the otapksPV gene, the influence of the temperature was system- atically analysed on laboratory media at high water activity values. For this reason P. verrucosum was grown for 5 days on YES medium at different temperatures. The RNA was extracted and subjected to Real Time PCR. In addition the ochratoxin A produced was determined by HPLC. The results are shown in Figure 4.

Table 2. HPLC data of the ochratoxin A concentration in the different wheat samples

Storage time [month]

moisture [%]

14

19

24

<LOD 1

<LOD

313+272

<LOD

<LOD

311+27

<LOD

<LOD

437+38

4

<LOD

<LOD

350+31

<LOD

<LOD

241+21

<LOD

<LOD

206+18

1LOD=Limit of Detection 2Average value of 4 independent experiments corrected for the water content, with its expanded uncertainty (k=2), the values are given in pg/kg

Table 3. Time course of the temperature during the storage of the wheat samples

Storage time [month]

moisture [%]

14

19

24

1the average values are given =n ~

211

20

21

20

19

21

11

11

11

4

9

8

9

142


Figure 3. Ochratoxin production kinetics (A) determined by HPLC and otapksPV expression determined by Real Time PCR (B)

Figure 4. Real Time expression data (dark columns) of the otapksPV gene and ochratoxin A determined by HPLC (light columns) of P. verrucosum grown for 5 days on YES medium at different temperatures

143


Interestingly two activation peaks arose, a major peak close to the growth optimum and a minor peak at much lower temperatures, close to the cessation of growth. These peaks could be seen at the phenotypical (ochratoxin A production) as well at the molecular level ( o t a p k s P V gene expression). This expression behaviour indicates higher ochratoxin A biosynthesis at lower and higher temperatures.

Discussion

The moisture content of stored wheat is of ut- most importance for its quality, as it influences growth and ochratoxin A biosynthesis of P. v e r r u c o s u m (15). A model of toxin formation in relation to moisture content and storage time is described (17). According to this model the start of ochratoxin formation depends on the moisture content of the wheat. At a moisture content of 24% ochratoxin A production starts within a few days according to this model. That is in good agreement with the results presented here. Already at the first time point, e.g. after a storage time of 4 weeks the maximum amount of ochratoxin A was produced. However in contrast no ochratoxin A was produced at storage conditions of 19% moisture content even after prolonged storage. According to the results of Gareis and R6del (18) a moisture content of_>l 9% is exactly the limit. They found ochratoxin A after a storage of barley for 2 weeks at a moisture content of 19%. Below these conditions P. v e r r u c o s u m is not able to produce the toxin. In another published model (17) ochratoxin A biosynthesis took place after a storage of 40 days at these conditions. We did not find ochratoxin A in the 19% sample, which might be due to the P. v e r r u c o s u m strain used in this study. Ob- viously this strain is not very resistant to draught stress. According to Cairns-Fuller et al.

(24) P. v e r r u c o s u m shows optimal growth conditions at a water activity of 0.98 which is quite high. In agreement with the generally described safe moisture level for wheat storage of about 14.5% (16, 17) we did not find any growth or ochratoxin A production activity at storage conditions of 14% moisture content, indicating that this conditions are indeed not suitable for metabolic activity of P. verru-

cosum.

Interestingly the cfu values increase over the whole observation period of 6 months from

about 10 7 to 10 9 cfu per gram in the 24% sample. In contrast the amount of ochratoxin A produced did not increase during this time but rather fluctuates around a certain average value. This indicates that the steady state level is already reached at the first time point after 4 weeks of storage. This fluctuation implies that there seems to be some kind of balance between production and degradation. This behaviour can also be observed in a pure P. ver-

r u c o s u m culture in a laboratory medium (data not shown), indicating that both activities, production and degradation, are due to P. verru-

c o s u m and not to certain environmental conditions. The phenotypic production of ochratoxin A can be visualized also at the molecular level by measuring the transcription of the o t a p k s P V gene. Also at this level a similar fluctuating behaviour could be observed. Interestingly after 3 month of storage (Novem- ber 06) a peak in the transcription of this gene was observed. This is paralleled by the highest ochratoxin A concentration in this sample. It has recently been shown that certain external stress factors like lowering the temperature or water activities to less optimal conditions or other stress factors can lead to activation of mycotoxin biosynthetic genes (25). In fact a detailed microarray analysis of the expression of ochratoxin A biosynthesis genes in P. nord icum, the other ochratoxin producing Pen ic i l l i um species, revealed a very high activity of some of the ochratoxin A biosynthesis genes at 15 ~ despite the fact that the growth optimum is between 20 ~ and 25 ~ (21). During the described experiments it could be shown that P. v e r r u c o s u m has two peaks of activation of ochratoxin biosynthetic genes, e.g. one major peak near the growth optimum at 25 ~ ~ and one minor peak at suboptimal growth conditions, e.g. at 15 ~ That fits very well with the observation made during the storage of the wheat. During the storage from month 2 (October 2006) to month 3 (November 2006) the ambient temperature dropped from around 20 ~ (which is below the optimum peak of activation) to 10 ~ That is about the temperature range at which the minor induction of the Pen ic i l l i um ochratoxin biosynthesis genes occurs. This influence of the temperature shift on induction of ochratoxin A biosynthetic genes would explain the high ochratoxin A biosynthesis activity during November 06. This is a novel feature of the regulation of ochratoxin A biosynthesis by P.

144


ver rucosum. Obviously in case when the moisture content is optimal for growth the temperature plays a pivotal role in regulation. Under these conditions very high amounts of ochratoxin A are produced at high temperatures (between 25 ~ ~ near the growth optimum, however a moderate induction of ochratoxin biosynthesis is also observed at temperatures which impose a stress to P. ver-

rucosum. In the range between these peaks (25 ~ ~ P. v e r r u c o s u m still produced ochratoxin A, however at a reduced rate. Under practical aspects high temperatures, as Well as temperatures which impose stress conditions to the fungus should be avoided as long as the moisture content is high. The congruence between the cfu data and the results of the molecular detection and monitoring systems demonstrate their usefulness to follow the fate of P. ve r rucosum. The fact that the sensitivity of the molecular methods ceases at cfu values below 103 per gram is known (19), as well as the fact that Real Time PCR data are usually above the cfu data, but after taking this into account these methods can be applied to rapidly assess the fungal status of a wheat sample.

Acknowledgement

We would like to thank Nicole Mischke, Doreen Roblick, Lars Uhlmann and Alexander Hanak for excellent technical assistance.

References

1 Petzinger E, Ziegler K (2000) Ochratoxin A from a toxicological perspective. J Vet Pharma- col Therap 23:91-98

2 European Commission (2006) Commission Re- gulation (EC) No 1886/2006 of 19 December 2006 setting maximum levels for certain con- taminants in foodstuffs. OJ L364:5-24

3 Lund F, Frisvad JC (2003) Penicillium verrucosum in wheat and barley indicates presence of ochratoxin A. J Appl Microbiol 95:1117-1123

4 Bucheli P, Kanchanomai C, Meyer I, Pittet A (2000) Development of Ochratoxin A during Robusta (Coffea canephora) Coffee Cherry Drying. J Agric Food Chem 48:1358-1362

5 Abarca ML, Accensi F, Bragulat MR, Castella G, Cabanes FJ (2003) Aspergillus carbonarius as the main source of ochratoxin A contamination in dried vine fruits from the Spanish market. J Food Protect 66:504-506

6 Thirumala-Devi K, Mayo MA, Reddy G, Emmanuel KE, Larondelle Y, Reddy DVR (2001) Occurrence of ochratoxin A in black pepper, coriander, ginger and turmeric in India. Food Addit Contam 18 (9): 830-835

7 Gareis M (1996) Fate of ochratoxin A on pro- cessing of meat products. Food Addit Contam 13:35-37

8 Mantle PG (1998) Ochratoxin A in Coffee. J Food Mycol 2:63-65

9 Frisvad J, Frank JM, Houbraken JAMP, Kuijpers AFA, Samson RA (2004) New ochratoxin A producing species o f Aspergillus section Circumdati. Stud Mycol 50:23-43

10 Varga J, Kozakiewicz Z (2006) Ochratoxin A in grapes and grape-derived products. Trends Food Sci Tech 17:72-81

11 Larsen TO, Svendsen A, Smedsgaard J (2001) Biochemical characterization of ochratoxin A- producing strains of the genus Penicillium. Appl Environ Microbiol 67:3630-3635

12 Castella G, Larsen TO, Cabanes J, Schmidt H, Alboresi A, Niessen L, F/~rber P Geisen R (2002) Molecular characterization of ochratoxin A producing strains of the genus Penicillium. System Appl Microbiol 25:74-83

13 Bogs C, Battilani P, Geisen R (2006) Develop- ment of a molecular detection and differentia- tion system for ochratoxin A producing Peni- cillium species and its application to analyse the occurrence of Penicillium nordicum in cured meats. Int J Food Microbiol 107:39-47

14 Cholmakov-Bodechtel C, Wolff J, Gareis M, Bresch H, Engel G, Majerus P, Rosner H, Schneider R (2000) Ochratoxin A: Represen- tative food consumption survey and epidemio- logical analysis. Arch Lebensmittelhyg 51: 111- 117

15 Lindblad M, Johnsson P, Jonsson N, Lindqvist R, Olsen M (2004) Predicting noncompliant levels of ochratoxin A in cereal grain from Penicillium verrucosum counts. J Appl Micro- biol 97:609-616

16 Olsen M, Jonsson N, Magan N, Banks J, Fanelli C, Rizzo A, Haikara A, Dobson A, Frisvad J, Holmes S, Olkku J, Persson S-J, B6rjesson T (2006) Prevention of ochratoxin A in cereals in Europe. In: Hocking AD, Samson RA, Pitt JI, Thrane U (eds) Advances in Food Mycology. Springer Verlag, Heidelberg, 317-342

17 Anonymous (2005) Home-Grown Cereals Authority, www.hgca.com

18 Gareis M, ROdel W (2002) Produktion von Ochratoxin A und B durch Penicillium verrucosum auf Gerste in Abh/ingigkeit der Para- meter Wasseraktivit/it, Wassergehalt und Tem- peratur. Mycotoxin Research 18A: 132-137

19 Schmidt-Heydt M, Richter W, Michulec M, Buttinger G, Geisen R (2007) A comprehensive molecular system to study presence growth and

145


ochratoxin A biosynthesis of P. verrucosum in wheat. Food Addit Contam (submitted)

20 Abramson D, Richter W, Rintelen J, Sinha RN, Schuster M (1992) Ochratoxin A production in Bavarian cereal grains stored at 15 and 19% moisture content. Arch Environ Contam Toxicol 23:259-265

21 Schmidt-Heydt M, Geisen R (2007) Gene expression as an indication for ochratoxin A biosynthesis in Penicillium nordicum. Mycotoxin Research 23: 13-21

22 Geisen R (2004) Molecular monitoring of environmental conditions influencing the induction of ochratoxin A biosynthetic genes in Penicillium nordicum. Mol Nutr Food Res 48: 532-540

23 European Committee for Standardization (2003) Foodstuffs - Determination of ochratoxin A in barley and roasted coffee - HPLC method with immunoaffinity column clean - up. European Standard EN 14132:2003

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

25 Schmidt-Heydt M, Baxter E, Geisen R, Magan N (2007A) Physiological relationship between food preservatives, environmental factors, ochratoxin and otapksPV gene expression by P. verrucosum. Int J Food Microbiol (accepted)

146

molecular and chemical monitoring of growth and ochratoxin a biosynthesis ofp. verrucosum in wheat...

Documents