Proceedings of the 7th International Worhng Conference on Stored-product Protectwn - Volume 1

Occurence of moulds and some mycotoxins in wheat importedin republic of macedonia

Manja Skrmjar", M. Danev", B. Petreskr", Manja Sanc1 and Biljana Cepreganova-Krstlc2

Abstract

The occurence of moulds and some mycotoxms (alfatoxmB1-AB1, B2-AB2, G1-AG1, G2-AG2, ochratoxm A-OA,zearalenone ZEA) m wheat Imported in Republic ofMacedonia was investigated. Wheat mtended for humanconsumption was Imported m penod January-March 1998.Direct platmg method was used to estimated the number

of moulds per wheat kernel Ouahtatrve and quantitativedetermmation of mycotoxms was carried out usmg the multi-mycotoxm methods descnbed by Scortichim et al. (1996).It was found that all of wheat samples tested (35) were

contaminated with moulds. The number of moulds rangedfrom 0.47 to 1.03 per kernel. Isolated fungi were classifiedmto 13 genera and 21 species as follows: Absidiacorumbifer«, Alternaria aliemata , A. chlamydospora,Arthrinium phaeospermum, A. sacchancola,Aspergtllus flavus , A. nuter , A versicolor,Cladosporiurn. spongiosum, Culoularia brachyspora,Drechslera state of Cochliobolus bicolor , D. state ofCochliobolus nodulosus, Euroiuum. herbariorurn ,Fusarium culmorum , F. lateritium , F. poae , F.proliferatum, Mucor chistumiensis , M. hiemalis ,Mycelm sierilui and. Peniculiura aurantwgriseumMoulds of Dematuiceous Hyphomycetes group were the

most frequent in Isolated mycopopulations Even 94 % ofwheat samples were infected WIth them. A alternarui as adommant mfectant was identified from 86 % samples ABl ,AB2, AGI and ZEA were not detected in wheat. But, 60%of samples contamed AG2 and OA at concentrations between0.5 and 1.0 flglkg.

Introduction

Cereals are a very good substrate for growth of mouldsSome of them are found on the surface of kernels assaprophrtes, while others penetrate the tissue and destroy Itby their metabolinc actrvity Such kernels are wnnkle ,

t Uruversttyof NOVl Sad, Faculty of Technology, 21000 NOVl Sad,

YugoslaVla;2 Umverstty 'Kmlt MetodtJe' , Vetermary Instttute and

3 Mtmstry of Health, 91000 SkOPJe, Repubhc of Macedoma

loose weight, surface becomes shmeless and the layer oftenchanges Its color. Decrease m technological quality ofdamaged kernels IS mevitable, which leads to great

economical losses.Many moulds which mfect wheat kernel in field or later,

dunng storage, are toxigenic and m suitable conditions mayform different toxic metabolites. Smce mycotoxins aremostly extracellular metabohtes, after synthesis theypenetrate mto a substrate, m this case m kernel tissue.Mycotoxms are very thermostabile compounds

Therefore, their structure IS destroyed only m part or not atall durmg bakmg of bread or other bakery products. Takmgmto consideration that they are extremely harmfull forhuman health, regular control of cereals, as well as otheragncultural products for presence of mycotoxms, is

necessary.Objective of this paper was to examme presence of moulds

WIth a special review on toxigenic species, as well as somemycotoxins m wheat intended for human consumption.

Material and methods

Presence of moulds and aflatoxms B1 (ABl), B2 (AB2) , G1(AG1), G2 (AG2), ochratoxm A (OA) and zearalenone(ZEA) , was exammed m 35 samples of wheat Imported fromSouth Amenca to the Republic of Macedonia, during thepenod from January to March 1998.

Mycological examinations

10 g were measured of each sample into an Erlenmayerbottle (300 ml ) and treated WIth 100 ml of 4%sodiumhypochlonde in order to remove all probably presentsaprophyte moulds from kernel surface Erlenmayer bottlewas mixed with the sample and sodiumchlonde on a rotatoryshaker, dunng 2 nun. After that, each sample was washedout WIth stenle , distilled water (2 x 100 ml) Under septiccondrtions, 10 kernels were placed on the surface of potatodextrose agar (PDA), addmg antibiotics (1 ml of 1%chloramphemcol and 1 ml of 1% oxytetracyclIne/100 ml ofmedium) Incubation of Petri dishes was carned out at25°C, dunng 7 days. The expenments were performed mtrIplIcates. Growth of moulds was monitored daIly. Theresults were read after 7 days and presented as an averagenumber of moulds per wheat kernel.

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Proceednl{Jsof the 7th Internatwnal Worhng Conference on Stored-product Protectwn - Volume I

Determination of Isolated species was earned outaccording to Ames (1969), Ellis (1971, 1976), Pidoph ckoand Miljko (1971), Nelson et al (1984) and Samson andvan Reen-Hoekstra (1988)

Mycotoxicological examinations

Quahtative and quantitative deternunation of mycotoxmswas performed applying a multunycotoxm procedure usmgsolid-phase extraction descnbed by Scortrchmi et al.(1996).

Results and discussion

Mycological examinations

Results of mycological examinations indicated that allkernels were mfected by moulds, although It could not beregistered visually for all wheat kernels. Accordmg to theappearance, some kernels were unhealthy and shnvelledDIscoloration was also registered. Therefore, the resultsobtamed, concernmg moulds m kernel tissue, were notunexpectedTable 1, concernmg number of moulds per kernel, shows

that all kernels were mfected by moulds. Average number ofmoulds ranged from 0.47 (sample num. 33) to 1.03(sample num. 20) per kernel.Moulds Isolated from the wheat samples exammed were

classified mto 13 genus and 21 species: Absidiacorumbifera (Cohn) Sacc & Trotter, Alternariaalternata (Fr.) Keissler , A. chlamydospora Moucchaca,Arthrmium phaeospermuni (Corda) M. BEllIs, A.saccharicola Stevenson, Asper[ftllus flavus Lmk , Aniger van Tieghem, A. oersicolor (VUIll ) Tirabosclu ,Chaetomsum globosum Kunze ex Fries, Chladosporiumsponqioeum. Berk & Curt, Culoularia brachysporaBoedijn, Drechslera state of Cochliobolus bicolor Paul &Parbery, D. state of Cochlwbolus nodulosus Luttrell,Eurotium. nerbomoruni (WIggers) Link, Mucorchristiamensis Hagem, M hiemalis Wehmer, Mycehaeterilia., Fusarium culmorum. (W. G. Smith) Sacc , F.laieruium. Nees, F poae (Peck) Wollenw, F.proliferatum. (Matsusluma ) NIrenberg and Penicillium.curomiuxmseurn. Dierckx.Moulds from group Dematuiceous Hyphomycetes (FIg.

1) had the greatest share m mycopopulations, as well asspecies from genus Fussarium. (FIg 2), which wereisolated from 94 and 80% of wheat samples, respectivelyThe most frequent among moulds from group DemaiuiceousHyphornycetes was A alternata, WhIChwas noticed to bemfectant m even 86% of the samples. A alter-nata ISknown for cosmopolitic spreadmg It IS considered a weekparasite and IS very often Isolated from cereals, differentfoodstufs and soil (Stinson et al 1980, PItt and Hockmg1985, Sknnjar et al. 1996.).

Table 1. Total number of moulds per wheat kernel.

Sample Total number of

No SIgn moulds per kernel

1 1/2 45790/12 o 822 13-4117-1 0.70

3 13-4117-2 0.88

4 13-4117-3 0.71

5 13-4117-4 0.57

6 13-4117-5 0.82

7 13- 4317-1 0.57

8 13- 4317- 2 0.90

9 13- 4317- 3 0.94

10 13-4417-1 0.87

11 13-4417 - 2 o 8712 13-4817- 2 0.77

13 13- 52/L-l 0.72

14 13- 52/L-2 0.77

15 13-5517-1 0.47

16 13- 5517- 2 0.87

17 13- 5617-1 10.1

18 13- 5617- 2 0.92

19 13- 5617- 3 0.57

20 13- 5617-4 1.03

21 13- 5617- 5 0.77

22 13- 5917-1 0.92

23 13-6817 - 2 0.82

24 13-6817 - 2 0.58

25 13- 6817- 2 0.60

26 13-6817-2 0.57

27 13-6917-2 0.75

28 1/1- 45778-1 0.62

29 III -1 0.70

30 III KAZ o 6731 IVKAZ 0.75

32 III SV 0.57

33 IV SV 0.47

34 VSV 0.70

35 VISV 0.57

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Proceedings of the 7th International Working Conference on Stored-product Protection - Volume 1

100908070605040302010o

c o E ~uldsA B

Fig. 1 Frequency of some moulds of Dematiaceous-Hyphomycetes group.

A - moulds., B - Fusarium spp.DC - F.culmorumCD - F.lateritium., E - F.poaeF - F.proliferatum

A - moulds

= 1000 90.- • B - Dematiaceous -.....ca= 80 Hyphomycetes group.-S 70 oC - Alternaria spp.cad 600c..>..... 50 CD - Drechslera spp.0IU 40onC'\S • E - Arthrinium spp...... 30=(l)0 205-c(l) F - Cladosporium sp.~ 10

0 • G - Culwlaria sp.A B C D E FMoulds

Fig. 2 Level of wheat contamination by Fusarium spp.

A. alternata has the ability to synthetisize differenttoxic metabolites, including alternariol, tenuzoic acid andalterotoxins (Stinson 1985, Stinson et al. 1982). Evenbefore structure of some toxins of A. alternata wasdetermined, Pero et al (1973) reported that there was anaccute toxicity in mice because of their action, while Scottand Stoltz (1980) pointed out mutagenic changes in cells ofSalnwnella typhimurium, also provoked by action ofthese toxins.Presence of Fusarium species in wheat kernels is of

particular importance, because they are toxigenic species.Fusarium species attack cereals already during the periodof vegetation causing deterioration of germs and youngplants, as well as seed damage. Some of the isolatedspecies, such as F. culmorum in adult plants causes

rotting of stem system and premature ripening, as well asoccurrence of white spike. During storage, kernels becomeinfected by mould and seeds germinate (Jovicevic andMilosevi t 1990). During the research, F. culmorum wasisolated from 51% of wheat (Fig. 2). This specie is widelyspread, particularly in temperate climatic regions. Amongmycotoxins produced by F. culmorum, the mostsignificant are trichothecenes and zearalenone (Marasas etal. 1984.).Other Fusarium species, also isolated from certain

wheat samples, were less spread. F. lateritium wasisolated from 28% of samples and F. poae and F.proliferatum from 11% and 9%, respectively. All threespecies were mycotoxigenic.

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Prcceedsnqs of the 7th Internatwnal Worktng Conference on Stored-product Protecium. - Volume 1

Mycotoxicological examinations

Non one of wheat sample contained ABl, AB2, AG1 orZEA (Table 2). However, among 15 samples examinated,even 9 (60 %) had AG2 in concentrations from O. 5 to 1. 0fLgikg. It could be interesting to point out that all wheatsamples, WhIChcontained AG2, were contaminated by AO atthe same time and relatively same concentrations.

Table 2. Contamination of wheat by alfatoxms, ochratoxin Aand zearalenone.

Samples AB1 AB2 AG1 AG2 OA ZEA

No SIgn

1 1/2 45790/122 13-41/7-1

13-41/7-213-41/7-313-41/7-413-41/7-5

3 13-43/7-113 -43/7-213 - 43/7 - 3

4 13-44/7-113-44/7 - 2

5 13-48/7- 26 13 - 52/L-1

13 - 52/L- 27 13 - 55/7-1

13 - 55/7 - 28 13 - 56/7-1

13-56/7 - 213- 56/7 - 313- 56/7-413 - 56/7- 5

9 13 - 59/7-110 13 - 68/7 - 2

13 - 68/7 - 213 - 68/7 - 213-68/7 - 2

11 13 -69/7-112 1/1- 45778-113 II-I14 III KAZ

IVKAZ15 III SV

IV SVVSVVI SV

0.5 1.0

0.5 1.0

0.5 1.00.5 1.0

0.8 0.5

0.5 0.5

0.5 1.00.8 1.0

0.5 1.0

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