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Aflatoxin production on wheat seed stored in air and nitrogen
Notes and brief articles RAPER, K. B. & FENNELL, D. I. (1965). The Genus Aspergillus. Baltimore: Williams and Wilkins. ROCKLAND, L. B. (1960). Saturated salt solutions for static control of relative humidity between 5° and 40°. Analytical Chemistry 3%, 1375-1376.
197
SNOW, D. (1949). The germination of mould spores at controlled humidities. Annals of Applied Biology 36, 3-13.
AFLATOXIN PRODUCTION ON WHEAT SEED STORED IN AIR AND NITROGEN A. A. FABBRI, C. FANELLI, M. SERAFINI, D. DI MAGGIO
Cattedra di Micologia, lstituto dell'Orto Botanico, Uniuersita di Roma, Largo Cristina di Svezia 2400165, Roma, Italy AND R. PIRAZZI
Encc (Centro Sperimentazione Agricola e Forestale), Casalotti, Roma
The contamination by aflatoxins of foods and feeds is a world problem, the economic and health consequences of which are so serious that it has become necessary to apply new methodologies to prevent, limit and eliminate their production (Austwick, 1975; Brekke et al., 1977). Modified atmospheres are one of the methods more widely used to prevent the growth of fungi producing toxins (Wilson & Jay, 1975; Epstein et al., 1970; Diener & Davis, 1969). High CO 2 or N 2 atmospheres with very low O 2 percentages have shown good results in limiting both production of toxins and growth of insects in silos of stored seeds (Jay & Pearman, 1973; Pearson & Sorensen, 1970). In Italy the problem of seed storage is very important, as foodstuffs are stored in silos for a long time before being used. In this work we have examined the production of aflatoxins on soft wheat seed stored in a N 2-controlled atmosphere with less than 0'03 % oxygen. The wheat used for this test presented moisture and temperature conditions favourable to the production of aflatoxins (Shih & Marth, 1974). The soft wheat seed utilized for experiments was sterilized by exposure to 60CO for 90 min at 6296 rad min-I using Gammacell 220 (Atomic Energy Ltd of Canada). After sterilization, seeds were moistened with sterile distilled water to a value of 18'5 % moisture content measured with a thermobalance (Buhler) and were inoculated with 8 x 106 conidia of Aspergillus fiavus Link ex Fr. (ATCC 22548) 200 g-I of seed. The wheat was put into 250 ern" cylindrical glass jars with a gas inlet in the upper part and an outlet in the lower part. The gas flowed from cylinders of N 2 (0'03 % O2) through a sterilizing filter (0'2 pm Gelman Preflow 200) and pressure regulators and micrometer regulating valves, connected to flowmeters, Trans. Br. mycol, Soc. 74 (1), (1980).
to regulate the flow to 4'5 ern" h- I. Triplicate jars in series were incubated at 32° in a thermostatically controlled incubator containing the same N 2 atmosphere. In a second series of jars a flow of air was substituted for N 2 • After 7, 14 and 21 days incubation, seed in three jars kept in N 2 and three in air were analysed for the production of aflatoxins. Samples (100 g) were prepared for extraction and quantitative analysis of aflatoxins by high-pressure liquid chromatography (HPLC) according to the method of Pons (1976). For purification of aflatoxins by column chromatography we have partially used the methods of Anon. (1975). Each lot of grain was homogenized using a Waring Blender and shaking for 30 min with 275 em" chloroform:water (10:1, vjv). The extracts were filtered through phase separation paper (Whatman, 1PS), concentrated to 1 em" on a rotary evaporator and purified by elution through a chromatographic column (30 x 200 mm) containing 10 g of silica gel 60 (Merck) supported on a thin layer of anhydrous sodium sulphate. The extracts in the column were eluted at a flow rate of 20 em" min- I with 150 em" hexane followed by 150 em" anhydrous ether. The adsorbed aflatoxins were eluted with 150 em" chloroform:methanol (19:1, vjv). This fraction was concentrated by evaporation to dryness and resuspended in 100 pI of the HPLC elution solvent, water-saturated chloroform : cycloexane : acetonitrile (50 : 15 : 2, v jv). For adjusting the retention time we used elution solvent: ethanol (98'5:1'5, vjv). HPLC separation was conducted with a Waters ALC-202 instrument equipped with M-6000 pump, H6K septumless injector and a small particle silica gel column (300 x 4 mm, stainless steel, packed with Lichrosorb, 10 pm) operating at 1800 psi with an elution rate of 2 ern" min-I. Aflatoxins were
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Notes and brief articles (0)
0·005
'"
0·004
::: :::
G,
"" ~
.D
~ <
0·003
.D
G,
0·002
0·00 \
4 T im e (min)
6
8
T im e (min)
Fig. 1. (A) High-pressure liquid chromatogram of aflatoxins standard B" B2, G" G. (50 ng each). Absorbance units full scale (365 nm) 0'01; (B) Chromatogram of the extract of wheat inoculated with A. ftavus grown 21 days in air atmosphere at 32 DC. Absorbance units full scale (365 nm) 0'01.
Table
1.
Aflatoxin content in wheat seeds stored in air and N. controlled atmospheres
Aflatoxin content (pg kg:") Incubation time (days) 7
14 21
BI
B.
,...-----A--,
~
Air 0'37 7'04 118'04
N.
Air
N,
0'45
0'35 0'75 5'00
0'28
REFERENCES
detected at 365 run on a colorimeter (Waters 440) coupled to a Hitachi chart recorder. Aflatoxin (Mackor Chemicals) standards were prepared in HPLC elution solvent and concentrations were determined by UV as described by Anon. (1975). Retention times of 4-6 min for aflatoxin standards (Fig. 1 a) were highly reproducible. Only aflatoxins B, and B. were found during the experiments (Table 1). Whilst aflatoxin levels increased between 7 and 21 days in the wheat stored in air, Trans. Br. mycol, Soc. 74 (1), (1980).
small amounts were detected only after 21 days in the N. atmosphere. In the enclosed conditions of the experiments, with less than 0'03 % O 2 both inside the jars and in the surrounding incubator, inhibition of aflatoxin production by high N 2 concentrations (Wilson & Jay, 1975) has been confirmed.
ANON. (1975). 12th ed. (ed. W. Horwitz), Washington: Benjamin Franklin Station, Association of Official Analytical Chemists. AUSTWICK, P. K. C. (1975). Mycotoxins. British Medical Bulletin 31, 222-229. BREKKE, O. L., SINNUBER, R. 0., PEPLINSKI, J. A., WALES, J. H., PUTNAM, G. B., LEE, D. J. & CIEGLER, A. (1977). Aflatoxins in corn: ammonia intactivation and bioassay with rainbow trout. Applied and Environmental Microbiology 34, 34-37. DIENER, U. L. & DAVIS, N. D. (1969). Production of aflatoxins on peanuts under controlled environments. Journal of Stored Products Research 5, 251-258. EpSTEIN, E., STEINBERG, M. P., NELSON, A. 1. & WEI,
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0007-1536/80/2828-5930 $00.35 © 1980 The British Mycological Society
Notes and briefarticles L. S. (1970). Aflatoxin production as affected by environmental conditions. Journal of Food Science 35, 389-391, JAY, E. G. & PEARMAN, G. C. (1973). Carbon dioxide for control of an insect infestation in stored corn (maize).Journalof Stored Products Research 9, 25-29. PEARSON, N. K. & SORENSEN, J. W. (1970). Use of gaseous nitrogen for controlling stored product insects in cereal grains. CerealChemistry 47,679-682. PONS, W. A. (1976). Mycotoxins:resolution of afla-
199
toxins Bu B., G 1, and G 2 by high-pressure liquid chromatography. Journal of the Association of Official Analytical Chemists 59, 101-109. SHIH, C. N. & MARTH, E. H. (1974). Some cultural conditions that control biosynthesis of lipid and aflatoxin by Aspergillus parasiticus, Applied Microbiology 27, 452-456. WILSON, D. M. & JAY, E. (1975). Influence of modified atmosphere storage on aflatoxin production in high moisture corn. Applied Microbiology 29, 224-228.
A BLIND-SEED DISEASE OF GLOBE ARTICHOKE A. L. J. COLE Department of Botany, University of Canterbury, Christchurch, New Zealand
While collecting seeds from the seed heads of globe artichoke (Cynara scolymus) it was noted that many seeds were easily split open by gentle compression. Within these seeds a single, large, nearly spherical, black sclerotium had replaced the entire contents of the seed (Fig. 1). The outer coat of the seed appeared normal in every respect and the infected seeds were indistinguishable from healthy seeds. This disease could be classified as a true blind-seed disease. In some seeds examined
from adjacent seed heads sclerotia were found on the seed coat and within the seed, having only partially replaced the endosperm. Sclerotia were germinated on sterile glass microscope slides in a moist chamber at 20°C. Hyphae developing from sclerotia were transferred to potato-dextrose agar plates and incubated at 25°. In all cases the resultant mycelial growth and sclerotium formation closely resembled that of Sclerotinia sclerotiorum (Lib.) DeBary.
OCCURRENCE OF BASIDIOMYCETES ON ARSENIC-TOXIC MINE WASTE L. M. BENSON, R. L. EVANS AND P. J. PETERSON Department of Botany and Biochemistry, Westfield College, University of London, Kidderpore Avenue, London NW3 7ST
Arsenic, together with copper and tin, have been mined and smelted in Devon and Cornwall, S.W. England, for several centuries. Several spoil heaps of the associated waste materials, containing high levels of arsenic, have remained undisturbed since the early twentieth century. Investigations into the biogeochemistry of arsenic, on a range of these derelict arsenic toxic sites, have been in progress over the past six years (Porter & Peterson, 1975, 1977a, b). Until the sitings reported here, no macro-fungi were observed on any of the sites. However, Pyatt (1973) observed sporophores of Laccaria laccata on a similar site. In 1977 and 1978, during regular visits to the spoil areas, fruit bodies of the following species were collected and identified from Gawton United Mine site (grid ref. 5 x 453688): Amanita muscaria (L. ex Fr.) Hooker; Lactarius rufus (Scop. ex Fr.) Fr.; Paxillus inuolutus (Batsch ex Fr.) Fr.; Trans. Br, mycol. Soc. 74 (1), (1980).
Scleroderma citrinum Pers.; Thelephora terrestris Ehrh. ex Fr.; Laccaria laccata (Scop. ex Fr.) Cooke; Leccinum scabrum (Bull. ex Fr.) S. F. Gray. No macro-fungi were found on any of the other sites visited in 1977 but isolated specimens of P. inoolutus, L. laccata and L. scabrum were recorded from other sites in 1978. The fruitbodies were growing directly out of the steep face of the spoil and smelter tip material and in places up to 6 m from the nearest green plants. Tree roots were not evident immediately below or near to fruitbodies though extensive digging was not carried out. The fungi were only present on the sides and base of the spoil which were otherwise uncolonized. The top of the tip was sparsely colonized by Agrostis tenuis, A. stolonifera and in parts by Calluna vulgaris. The site was surrounded by woodland comprising Betula pubescens and Quercus species with a ground
Printed in Great Britain
0007-1536/80/2828-5930 $00.35 © 1980 The British Mycological Society
197
SNOW, D. (1949). The germination of mould spores at controlled humidities. Annals of Applied Biology 36, 3-13.
AFLATOXIN PRODUCTION ON WHEAT SEED STORED IN AIR AND NITROGEN A. A. FABBRI, C. FANELLI, M. SERAFINI, D. DI MAGGIO
Cattedra di Micologia, lstituto dell'Orto Botanico, Uniuersita di Roma, Largo Cristina di Svezia 2400165, Roma, Italy AND R. PIRAZZI
Encc (Centro Sperimentazione Agricola e Forestale), Casalotti, Roma
The contamination by aflatoxins of foods and feeds is a world problem, the economic and health consequences of which are so serious that it has become necessary to apply new methodologies to prevent, limit and eliminate their production (Austwick, 1975; Brekke et al., 1977). Modified atmospheres are one of the methods more widely used to prevent the growth of fungi producing toxins (Wilson & Jay, 1975; Epstein et al., 1970; Diener & Davis, 1969). High CO 2 or N 2 atmospheres with very low O 2 percentages have shown good results in limiting both production of toxins and growth of insects in silos of stored seeds (Jay & Pearman, 1973; Pearson & Sorensen, 1970). In Italy the problem of seed storage is very important, as foodstuffs are stored in silos for a long time before being used. In this work we have examined the production of aflatoxins on soft wheat seed stored in a N 2-controlled atmosphere with less than 0'03 % oxygen. The wheat used for this test presented moisture and temperature conditions favourable to the production of aflatoxins (Shih & Marth, 1974). The soft wheat seed utilized for experiments was sterilized by exposure to 60CO for 90 min at 6296 rad min-I using Gammacell 220 (Atomic Energy Ltd of Canada). After sterilization, seeds were moistened with sterile distilled water to a value of 18'5 % moisture content measured with a thermobalance (Buhler) and were inoculated with 8 x 106 conidia of Aspergillus fiavus Link ex Fr. (ATCC 22548) 200 g-I of seed. The wheat was put into 250 ern" cylindrical glass jars with a gas inlet in the upper part and an outlet in the lower part. The gas flowed from cylinders of N 2 (0'03 % O2) through a sterilizing filter (0'2 pm Gelman Preflow 200) and pressure regulators and micrometer regulating valves, connected to flowmeters, Trans. Br. mycol, Soc. 74 (1), (1980).
to regulate the flow to 4'5 ern" h- I. Triplicate jars in series were incubated at 32° in a thermostatically controlled incubator containing the same N 2 atmosphere. In a second series of jars a flow of air was substituted for N 2 • After 7, 14 and 21 days incubation, seed in three jars kept in N 2 and three in air were analysed for the production of aflatoxins. Samples (100 g) were prepared for extraction and quantitative analysis of aflatoxins by high-pressure liquid chromatography (HPLC) according to the method of Pons (1976). For purification of aflatoxins by column chromatography we have partially used the methods of Anon. (1975). Each lot of grain was homogenized using a Waring Blender and shaking for 30 min with 275 em" chloroform:water (10:1, vjv). The extracts were filtered through phase separation paper (Whatman, 1PS), concentrated to 1 em" on a rotary evaporator and purified by elution through a chromatographic column (30 x 200 mm) containing 10 g of silica gel 60 (Merck) supported on a thin layer of anhydrous sodium sulphate. The extracts in the column were eluted at a flow rate of 20 em" min- I with 150 em" hexane followed by 150 em" anhydrous ether. The adsorbed aflatoxins were eluted with 150 em" chloroform:methanol (19:1, vjv). This fraction was concentrated by evaporation to dryness and resuspended in 100 pI of the HPLC elution solvent, water-saturated chloroform : cycloexane : acetonitrile (50 : 15 : 2, v jv). For adjusting the retention time we used elution solvent: ethanol (98'5:1'5, vjv). HPLC separation was conducted with a Waters ALC-202 instrument equipped with M-6000 pump, H6K septumless injector and a small particle silica gel column (300 x 4 mm, stainless steel, packed with Lichrosorb, 10 pm) operating at 1800 psi with an elution rate of 2 ern" min-I. Aflatoxins were
Printed in Great Britain
0007-1536/80/2828-5930 $00.35 © 1980 The British Mycological Society
Notes and brief articles (0)
0·005
'"
0·004
::: :::
G,
"" ~
.D
~ <
0·003
.D
G,
0·002
0·00 \
4 T im e (min)
6
8
T im e (min)
Fig. 1. (A) High-pressure liquid chromatogram of aflatoxins standard B" B2, G" G. (50 ng each). Absorbance units full scale (365 nm) 0'01; (B) Chromatogram of the extract of wheat inoculated with A. ftavus grown 21 days in air atmosphere at 32 DC. Absorbance units full scale (365 nm) 0'01.
Table
1.
Aflatoxin content in wheat seeds stored in air and N. controlled atmospheres
Aflatoxin content (pg kg:") Incubation time (days) 7
14 21
BI
B.
,...-----A--,
~
Air 0'37 7'04 118'04
N.
Air
N,
0'45
0'35 0'75 5'00
0'28
REFERENCES
detected at 365 run on a colorimeter (Waters 440) coupled to a Hitachi chart recorder. Aflatoxin (Mackor Chemicals) standards were prepared in HPLC elution solvent and concentrations were determined by UV as described by Anon. (1975). Retention times of 4-6 min for aflatoxin standards (Fig. 1 a) were highly reproducible. Only aflatoxins B, and B. were found during the experiments (Table 1). Whilst aflatoxin levels increased between 7 and 21 days in the wheat stored in air, Trans. Br. mycol, Soc. 74 (1), (1980).
small amounts were detected only after 21 days in the N. atmosphere. In the enclosed conditions of the experiments, with less than 0'03 % O 2 both inside the jars and in the surrounding incubator, inhibition of aflatoxin production by high N 2 concentrations (Wilson & Jay, 1975) has been confirmed.
ANON. (1975). 12th ed. (ed. W. Horwitz), Washington: Benjamin Franklin Station, Association of Official Analytical Chemists. AUSTWICK, P. K. C. (1975). Mycotoxins. British Medical Bulletin 31, 222-229. BREKKE, O. L., SINNUBER, R. 0., PEPLINSKI, J. A., WALES, J. H., PUTNAM, G. B., LEE, D. J. & CIEGLER, A. (1977). Aflatoxins in corn: ammonia intactivation and bioassay with rainbow trout. Applied and Environmental Microbiology 34, 34-37. DIENER, U. L. & DAVIS, N. D. (1969). Production of aflatoxins on peanuts under controlled environments. Journal of Stored Products Research 5, 251-258. EpSTEIN, E., STEINBERG, M. P., NELSON, A. 1. & WEI,
Printed in Great Britain
0007-1536/80/2828-5930 $00.35 © 1980 The British Mycological Society
Notes and briefarticles L. S. (1970). Aflatoxin production as affected by environmental conditions. Journal of Food Science 35, 389-391, JAY, E. G. & PEARMAN, G. C. (1973). Carbon dioxide for control of an insect infestation in stored corn (maize).Journalof Stored Products Research 9, 25-29. PEARSON, N. K. & SORENSEN, J. W. (1970). Use of gaseous nitrogen for controlling stored product insects in cereal grains. CerealChemistry 47,679-682. PONS, W. A. (1976). Mycotoxins:resolution of afla-
199
toxins Bu B., G 1, and G 2 by high-pressure liquid chromatography. Journal of the Association of Official Analytical Chemists 59, 101-109. SHIH, C. N. & MARTH, E. H. (1974). Some cultural conditions that control biosynthesis of lipid and aflatoxin by Aspergillus parasiticus, Applied Microbiology 27, 452-456. WILSON, D. M. & JAY, E. (1975). Influence of modified atmosphere storage on aflatoxin production in high moisture corn. Applied Microbiology 29, 224-228.
A BLIND-SEED DISEASE OF GLOBE ARTICHOKE A. L. J. COLE Department of Botany, University of Canterbury, Christchurch, New Zealand
While collecting seeds from the seed heads of globe artichoke (Cynara scolymus) it was noted that many seeds were easily split open by gentle compression. Within these seeds a single, large, nearly spherical, black sclerotium had replaced the entire contents of the seed (Fig. 1). The outer coat of the seed appeared normal in every respect and the infected seeds were indistinguishable from healthy seeds. This disease could be classified as a true blind-seed disease. In some seeds examined
from adjacent seed heads sclerotia were found on the seed coat and within the seed, having only partially replaced the endosperm. Sclerotia were germinated on sterile glass microscope slides in a moist chamber at 20°C. Hyphae developing from sclerotia were transferred to potato-dextrose agar plates and incubated at 25°. In all cases the resultant mycelial growth and sclerotium formation closely resembled that of Sclerotinia sclerotiorum (Lib.) DeBary.
OCCURRENCE OF BASIDIOMYCETES ON ARSENIC-TOXIC MINE WASTE L. M. BENSON, R. L. EVANS AND P. J. PETERSON Department of Botany and Biochemistry, Westfield College, University of London, Kidderpore Avenue, London NW3 7ST
Arsenic, together with copper and tin, have been mined and smelted in Devon and Cornwall, S.W. England, for several centuries. Several spoil heaps of the associated waste materials, containing high levels of arsenic, have remained undisturbed since the early twentieth century. Investigations into the biogeochemistry of arsenic, on a range of these derelict arsenic toxic sites, have been in progress over the past six years (Porter & Peterson, 1975, 1977a, b). Until the sitings reported here, no macro-fungi were observed on any of the sites. However, Pyatt (1973) observed sporophores of Laccaria laccata on a similar site. In 1977 and 1978, during regular visits to the spoil areas, fruit bodies of the following species were collected and identified from Gawton United Mine site (grid ref. 5 x 453688): Amanita muscaria (L. ex Fr.) Hooker; Lactarius rufus (Scop. ex Fr.) Fr.; Paxillus inuolutus (Batsch ex Fr.) Fr.; Trans. Br, mycol. Soc. 74 (1), (1980).
Scleroderma citrinum Pers.; Thelephora terrestris Ehrh. ex Fr.; Laccaria laccata (Scop. ex Fr.) Cooke; Leccinum scabrum (Bull. ex Fr.) S. F. Gray. No macro-fungi were found on any of the other sites visited in 1977 but isolated specimens of P. inoolutus, L. laccata and L. scabrum were recorded from other sites in 1978. The fruitbodies were growing directly out of the steep face of the spoil and smelter tip material and in places up to 6 m from the nearest green plants. Tree roots were not evident immediately below or near to fruitbodies though extensive digging was not carried out. The fungi were only present on the sides and base of the spoil which were otherwise uncolonized. The top of the tip was sparsely colonized by Agrostis tenuis, A. stolonifera and in parts by Calluna vulgaris. The site was surrounded by woodland comprising Betula pubescens and Quercus species with a ground
Printed in Great Britain
0007-1536/80/2828-5930 $00.35 © 1980 The British Mycological Society