Role of Aspergillus species in mycotoxin contamination

of agricultural products in Central Europe

Gyöngyi Szigeti, Baranyi, N., Kocsubé, S., Győri, T.,

Szekeres, A., Tóth, B., Török, O., Háfra, E., Pálfi, X.,

Varga, J.

Belgrade, 2012. 09. 27.

Aspergillus

•A common genus with about 300 species

•Calmodulin and β-tubulin sequences

•Preference for warmer climates

•Food spoilage organisms, mycotoxin

producers and opportunistic human

pathogens

•Production of enzymes and organic acids

•Pre- and postharvest contamination

•Genome sequences of 12 species are

known

Most important mycotoxins

produced by Aspergilli

• Aflatoxins

• Ochratoxins, citrinin

• Fumonisins

• Patulin

• Cyclopiazonic acid

• Gliotoxin

• Citreoviridin

Aflatoxins

Discovered in the 1960s (England)

Decaketide derivatives

Main producers: Aspergillus flavus, A. parasiticus, A. nomius

(A. ochraceoroseus, A. bombycis, A. pseudotamarii)

Occurrence: (mainly in tropical countries)

Peanut, tree nuts

Cotton

Spices

Rice

Cereals (corn)

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Aflatoxin B1

Effects of aflatoxins

Treated as „carcinogenic to humans” (IARC group 1)

Acute toxic hepatitis

1974, India (108 deaths from contaminated corn)

1981, 2004, 2006, Kenya (>150 deaths)

Chronic aflatoxicosis

Cirrhosis, liver cancer

High incidence in Mozambique, South Africa, China, Thailand, Philippines

Aflatoxins and potential aflatoxin producers

in agricultural products in Central Europe

Northern Italy: 2003: Identification of A. flavus and aflatoxin contamination exceeding the

EU limit in maize (Giorni et al. 2007)

Romania: 1997: all maize samples were negative for aflatoxins (Curtui et al. 1998)

2002-2004: 30% of the examined maize samples were contaminated, 20% of them above the EU limit (Tabuc et al. 2009)

Serbia: Aflatoxins have been detected in 18.3% of the examined maize samples

(Jakic-Dimic at al. 2009)

41.2% of the examined maize samples were contaminated with afltatoxins (Jaksic at al. 2011)

Aflatoxin M1 contamination exceeding the EU limit in 30.4% of milk samples (Polovinski-Horvatovic et al. 2009)

Slovenia: Aflatoxin M1 contamination exceeding the EU limit in 10% of milk

samples (Torkar & Vengust 2007)

Aflatoxins and potential aflatoxin producers

in agricultural products in Central Europe

Croatia: A. flavus was isolated from 38% of the flour samples (Halt et al. 2004)

Aflatoxin M1 in milk samples above the EU limit (Bilandzic et al. Markov et al. 2010)

Bulgaria: 40.95% of the examined wheat and maize samples were contaminated by

A. flavus and aflatoxins were detected in 65% of them above the EU limit (Borisova et al. 2008)

Hungary: 1992: none of the examined 22 A. flavus isolates from various sources

were able to produce aflatoxins (Richard et al. 1992)

Aflatoxin B1 levels above the EU limit in 4.8% of the examined cereal samples (Borbély et al. 2010)

64% of the examined maize samples were contaminated by A. flavus, and 42.4% of them were able to produce aflatoxins, however none of the samples contained aflatoxins above the EU limit (Dobolyi et al. 2011)

Occurrence of aflatoxins in Central

European countries

Identification of potential aflatoxin

producers in cereals in Hungary

Species assignment was based on sequence analysis of

part of the calmodulin gene

All of the isolates belong to A. flavus species

Proportions of the infected samples:

Maize: 0.83%

Wheat: 3.17%

Barley: 2%

None of the samples were contaminated by aflatoxins

25% of the isolates were able to produce aflatoxins B1

and B2

Aflatoxins produced by an A. flavus

isolate came from cereal

Ochratoxin A

Discovered in 1965

Pentaketide derivative

Main producers: Section Circumdati (A. ochraceus, A.

westerdijkiae, A. steynii), Flavi (A. albertensis), Nigri (A.

carbonarius, A. niger)

Nephrotoxic, immunosuppressant, teratogenic, carcinogenic

Chronic interstitial nephritis (Tunis)

Urothelial tumors (Egypt, Serbia, Croatia)

Renal failure due to acute tubular necrosis (Italy)

Balkan endemic nephropathy (?)

Treated as „possibly carcinogenic to humans” (IARC group 2B)

Occurrence of ochratoxins

• Cereals (wheat, corn, rice)

• Coffee, cocoa

• Spices (paprika, curry)

• Fruits and vegetables

• Wine, raisins, beer

• Pulses (soybean)

• Oil seeds (peanut)

• Dairy products

Mycobiota of grapes in Hungary in

2004-2006

Acta Aliment 36(2007)329–341

Ochratoxin A in Hungarian wines

Acta Aliment 36(2007)329–341

Detection of ochratoxin producing

Aspergilli on cereals in Hungary

• Maize: A. niger, A. awamori and A. tubingensis were identified

• Wheat: only A. niger was found

• Barley, rye: A. niger was detected

Fumonisins

Discovered in 1988 (South Africa)

Main producer: Fusarium verticillioides (endophyte of corn)

Polyketide derived mycotoxins

Sphinganine-analog structure

Leucoencephalomalacia in horses; Pulmonary edema in pigs;

Hepatocarcinoma in mouse, rat

Inhibit folic acid transport (Neural tube defects )

Associated with espophageal cancer

Treated as “possibly carcinogenic to humans” (IARC group 2B)

Fumonisin gene cluster in the genome of A. niger

Identified in A. niger strains CBS 513.88, ATCC 1015, N400, NRRL 3

Pel et al. Nat Biotechnol 25(2007)221-231; Baker Med Mycol 44(2006)S17-S21

97

95

100

76

99

87

96

99

100

72

7495

83

100

99

97

80

100

20

A. brasiliensis CBS 101740

A. carbonarius CBS 111.26

A. sclerotioniger CBS 115572

A. sclerotiicarbonarius CBS 121057

A. ibericus CBS 121593

A. piperis CBS 112811

A. acidus CBS 564.65

A. coreanus CBS 119384

A. tubingensis CBS 134.48

A. awamori CBS 557.65

A. niger CBS 554.65

A. foetidus CBS 114.49

A. foetidus CBS 121.28

A. lacticoffeatus CBS 101883

A. ellipticus CBS 707.79

A. heteromorphus CBS 117.55

A. homomorphus CBS 101889

A. fijiensis CBS 313.89

A. fijiensis CBS 119.49

A. aculeatinus CBS 1212060

A. aculeatus CBS 172.66

A. uvarum CBS 121591

A. indologenus CBS 114.80

A. japonicus CBS 114.51

A. violaceofuscus mut. grisea CBS 122.35

A. violaceofuscus CBS 123.27

A. violaceofuscus CBS 102.23

A. bahamensis CBS 115571

A. flavus CBS 100927

A. neoniger CBS 115656

A. neoniger CBS 115657

A. eucalypticola CBS 122712

A. vadensis CBS 113365

A. costaricaensis CBS 115574

A. aculeatus clade

A. carbonarius clade

A. homomorphus clade

A. heteromorphus clade

A. niger clade

Tree based on

calmodulin

sequence data

Varga et al. Stud Mycol (in press)

11/13 (84.6%) of the samples were heavily contaminated by black Aspergilli

Mycobiota of raisin samples

Fumonisin isomers in an A. awamori

isolate came from raisins

RP-HPLC-ESI-IT-MS

Fumonisins and black Aspergilli in

onions

Phylogenetic

tree based on

calmodulin

sequence data

Presence of fumonisins in onion

samples

~0.3 mg/kg fumonisins found in onions

Occurrence of Aspergilli on

spices and walnut

Potential mycotoxin producing species: A. flavus,

A. westerdijkiae, A. melleus, A. terreus, A.

awamori, A. niger

A. eucalypticola and A. amoenus were detected for

the first time in Europe

Examination of the mycotoxin producing abilities

and genetic variability of these species are in

progress

Summary

Because of the global warming a shift has recently been observed in the occurrence of aflatoxin producers in Europe

Our data confirm that aflatoxin producing A. flavus is present in Hungarian maize fields, similarly to that observed in neighboring countries

Several ochratoxin producing black Aspergillus species were also identified from grapes and cereals

The fumonisin producing A. awamori species was isolated from onions and fumonisins were detectable in some samples

Potential mycotoxin producers were isolated from spices and nuts

A. eucalypticola and A. amoenus species were detected for the first time in Europe

Further studies are in progress to examine the mycotoxin producing abilities of the isolates, and to examine the mycotoxin content of the samples

Acknowledgment

This work was supported by OTKA grant Nos. K84122 and

K84077, and by the János Bolyai Research Scholarship of the

Hungarian Academy of Sciences (B. Tóth).

The project is co-financed by the European Union through the

Hungary-Serbia IPA Cross-border Co-operation Programme

(ToxFreeFeed, HU-SRB/1002/122/062).

Thank you for your attention!