Penitrem and Thomitrem Formation by Penicillium Crustosum

Mycopathologia 157: 349–357, 2004. 349 © 2004 Kluwer Academic Publishers. Printed in the Netherlands. Penitrem and thomitrem formation by Penicillium crustosum Thomas Rundberget1, Ida Skaar1, Oloff O’Brien2 & Arne Flåøyen1 1National Veterinary Institute, PO Box 8156 Dep., 0033 Oslo, Norway; 2ARC Plant Protection, Research Institute, Private Bag X134, Pretoria 0001, South Africa Received 9 September 2002; accepted in final form 16 July 2003 Abstract The levels of penitrems A, B, C, D, E, F, roquefortine C and thomitrem A and E recovered from extracts of 36 Norwegian, 2 American and one each of Japanese, German, South African, Danish and Fijian isolates of Penicillum crustosum Thom were quantitatively determined using high performance liquid chromatography-mass spectrometry (HPLC-MS). Forty-two of the 44 isolates of penitrem-producing isolates grown on rice, afforded levels of thomitrem A and E comparable to that of penitrem A. Thomitrems A and E were also found, but at lower levels, when cultures were grown on barley. No thomitrems were found when the isolates were grown on liquid media. The effects of time and temperature on mycotoxin formation were studied on rice over a 4 week period at 10, 15 and 25 ◦C, respectively. No mycotoxins could be detected after 1 week at 10 ◦C, but after 2 weeks at 10 ◦C levels were similar to those produced at 15 and 25 ◦C. Higher levels of thomitrems A and E were detected when media were maintained at lower pH. The possibility that thomitrems A and E might be derived by acid promoted conversion of penitrems A and E was explored in stability trials performed at pH 2, 3, 4, 5 and 7 in the presence and absence of media. Thomitrems were formed at pH 2, 3 and 4 but not at pH 5 and 7. Key words: Penicillium crustosum, penitrems, thomitrem, foods, feeds Introduction thomitrems, prompted us to quantatively examine the extracts of 36 Norwegian together with one Danish, We have recently reported the occurrence of two novel one Fijian one Japanese, one Russian, one German, penitrem metabolites, thomitrems A and E, along with two American and one South African isolate of P. penitrems A, B, C, D, E and F from a single Norwe- crustosum available in our laboratory in order to gian Pencillium crustosum isolate 1590P2 grown on a ascertain the generality, or otherwise, of our earlier solid rice medium [1]. The detection of thomitrems findings. Additionally we also wanted to determine A and E, which unlike other known penitrem ana- the effects of solid and liquid media, pH, temperature logues, possess an 18(19)-double bond (Figure 1) is and incubation time on the formation of penitrems of interest in that so far the use of liquid cultures of and thomitrems under the condition utilized in our P. c ru sto su m have been reported to only afford roque- solid culture medium experiments since temperature, fortine C, penitrems A–F [2-3] and derivatives such water activity, medium and incubation time are known as penitremones [4], PC-M4, M5, M5 and M6 [5– to be important factors affecting fungal growth and 6]. Mycotoxins produced by P. c ru sto su m have been mycotoxin production [10]. reported to cause toxicity [7–9] in animals. Whether the thomitrems may contribute to P. c ru sto su m toxicity Materials and methods has not been studied. The unexpected occurrence of the thomitrems A Penicillium strains and E in the extracts of the 1590P2 isolate [1], and our qualitative observation that some other Norwe- Thirty-six Norwegian isolates of P. c ru sto su m from gian P. c ru sto su m isolates also appeared to produce Norwegian food and food waste together with one 350 added to a 50 ml Erlenmeyer flask and 0.1 M HCl was added dropwise to give an initial media pH of 2.5, 4, 5.5 and 7. The media were sterilised by autoclaving for 15 min. All media were point inoculated from malt extract agar [11] cultures incubated for 6 days at 25 ◦C. Incubation All of the P. c ru sto su m isolates were point inoculated and incubated on rice for 2 weeks at 20 ◦C. Four selec- ted penitrem producing Norwegian isolates, 1172F10, 1574F8, 1590P5 and 1592P5 (National Veterinary In- stitute numbers) were point inoculated and incubated on barley and on CDYE for 2 weeks at 20 ◦C. A Nor- wegian strain, 1590P5, and the South African strain, PPRI6859, were point inoculated and incubated on rice for 1, 2, 3 and 4 weeks at 10, 15 and 25 ◦C. There were three replicates of each treatment. Another Norwegian strain, 1564F1, was point inoculated and incubated for 1 week at 25 ◦C on liquid CYA media in which the pH had initially been adjusted to 2.5, 4, 5.5 and 7. PH stability trials Trial 1: The stability of penitrem A in acid was invest- igated by treating an acetonitrile:water (2:1) solution Figure 1. Chemical structure of thomitrem A, E, penitrem A and E. containing 100 ng/ml penitrem A with 0.01 M HCl (pH 2). The acidic penitrem A solution was kept at room temperature (20 ◦C)andanalysedbyhighper- Danish (IBN19484, Denmark Technical University, formance liquid chromatographic-mass spectrometry Copenhagen), one Fijian (CBS 313.48), one Japan- (HPLC-MS) every other hour for 24 h. ese (CBS 340.59), one Russian (CBS 499.73), one Trail 2: A one week old CYA inoculate (10 ml) German (CBS 483.75), two American (CBS 747.74 was diluted with 20 ml acetonitrile and placed in an and CBS 548.77) and one South African isolate ultra sonic bath for 10 min. The sample extract were (PPR15859, ARC Plant Protection Research Institute, filtered and the pH was adjusted to 3, 4 and 5 with Pretoria) were studied. 0.1 M HCl. The levels of penitrems and thomitrems were monitored at 12 h intervals for 72 h. The sample Media extracts was kept at room temperature (20 ◦C) between analysis. Rica and Barley: Twenty g of Uncle Ben’s rice and 60 ml distilled water were added to 500 ml Erlenmeyer Chemicals flasks and 20 g of unhusked barley and 12 ml distilled water were added to 250 ml Erlenmeyer flasks and Methanol, ethyl acetate, acetonitrile, hexane, am- sterilised by autoclaving for 15 min. monium acetate and formic acid were HPLC grade or Liquid media: Twenty ml of Czapek Dox yeast ex- p.a. grade obtained from Ratburn Chemicals (Walker- tract broth (CDYE) [12] without agar were added to a burn, U.K.). The Penicillium toxin standards roque- 100 ml Erlenmeyer flasks. The media were sterilised fortine C, and penitrem A were obtained from Sigma by autoclaving for 15 min. (St. Louis, USA), thomitrems A and E were isolated pH adjusted media: Ten ml of Czapek yeast (auto- at the National Veterinary Institute (Oslo, Norway), lysate) extract broth (CYA) [11] without agar were while penitrems B, C, D, E and F were gifts from 351 AgResearch Ruakura (Hamilton, New Zealand) and scan positive ion mode during the analysis. The ion Imperial College of Science Technology and Medicine injection time was set to 300 ms with a total of 3 (London, UK). micro-scans per second. For APCI a vaporisation temperature at 350 ◦C, a sheath gas rate at 25 units Extraction and clean up nitrogen (c 250 ml/min), an auxiliary gas rate at 5 units nitrogen (c 50 ml/min), a corona discharge voltage of Rice and barley cultures 4.5 V and a capillary temperature of 200 ◦Cwereused. The extraction procedure was based on a method described by Rundberget and Wilkins [13]. Each of the Screening for metabolites rice or barley culture flasks were extracted with 100 ml acetonitrile:water (9:1) for 5 min on an Ultrathurax Quantitative determination of penitrem A, roque- (Janke and Kunkel, Staufen, Germany). After de- fortine C and semi-quantitative determination of fatting with 50 ml of hexane, the extract was filtered penitrems B, C, D, E and F and thomitrems A and 1 E was performed using HPLC-MS. Semi-quantitative (Schleicher & Schuell 520 B 2 folded filters, Dassel, Germany). This extract was filtered further with a analyses were performed using a unit response (peak spin-X centrifuge filter (Costar, Corning, USA) and area) relative to penitrem A. Solid media (rice and then analysed on the described HPLC-MS system barley) results are reported as µg metabolite/g wet without further clean up. weight, while CDYE results are reported as µg metabolite/g liquid. CDYE cultures Cultures were extracted by shaking with 50 ml ethyl Results acetate for 10 min. After filtration, 10 ml of the ethyl acetate fraction was evaporated to dryness at 60 ◦C Screening of P. crustosum isolates under a gentle stream of nitrogen. The residue was redissolved in 900 ml acetonitrile and diluted with 100 Except for the Fijian and the Japanese strains, which ml water prior to HPLC-MS analyses. grew slowly, the other stains grew and sporulated vigorously after incubation on rice at 20 ◦C for 14 pH adjusted CYA cultures days. All stains were found to produce penitrems, ex- The CYA inoculate (10 ml) was diluted with 20 ml cept for 2 Norwegian isolates, which did not produce acetonitrile and placed in an ultrasonic bath for 10 penitrems. Among the penitrem producing isolates, min. The extract was filtered with a spin-X centrifuge thomitrems A and E, penitrem A and roquefortine C filter and then analysed on the described HPLC-MS were found to be major metabolites together with 2 system without further clean up.

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