Dario Ivić

Fumonisin B1, fumonisin B2, zearalenone and ochratoxin A contamination of maize in Croatia.

Mycotoxins are products of moulds that frequently contaminate maize. In this study the presence of mycotoxins fumonisin B1 (FB1), fumonisin B2 (FB2), zearalenone (ZEA) and ochratoxin A (OTA) was determined in 49 maize grain samples collected in autumn 2002. The most frequent finding was that of FB1(100%), followed by ZEA (84%) and OTA (39%), while FB2 was found only in three samples. The co-occurrence of two and three mycotoxins was found in 55 and 37% of samples, respectively. The concentrations (mean ± SD) of FB1, ZEA and OTA in positive samples were 459.8 ± 310.7, 3.84 ± 6.68 and 1.47 ± 0.38 µg kg−1, respectively, and the concentrations of FB2 in three positive samples were 68.4, 109.2 and 3084.0 µg kg−1. Although such low concentrations of mycotoxins are not a significant source of exposure in countries with a European diet, a few samples with extreme values indicate that thorough control is needed.

Ochratoxin A Contamination of Food from Croatia

Ochratoxin A (OTA) is a mycotoxin with nephrotoxic, genotoxic and carcinogenic properties produced by Penicillium and Aspergillus moulds under different climatic conditions. Humans and animals are exposed to this compound mainly via ingestion of contaminated food. In Croatia, research on mycotoxins focused on OTA when the mycotoxin theory of endemic nephropathy (EN) was postulated. Ochratoxin A was more frequent and at higher concentration in foods from EN than those from the control regions. Subsequently, OTA concentrations were determined in some commodities intended for human consumption such as maize, wheat, beans and wine. Samples from all parts of Croatia were analyzed and OTA was found in all types of commodities. It was frequently found together with other mycotoxins (fumonisin B1, fumonisin B2 and zearalenone). In general, OTA concentration in foods from Croatia is low, but the frequency of positive samples shows considerable variations from year to year depending also on sampling location. Although low levels of OTA were found in a large proportion of analyzed food samples, its persistent co-occurrence with other significant mycotoxins should raise serious public health concerns as there interactions may be synergistic or additive in causing toxicity in humans and animals. There is need to establish control measures through which such contaminations in foods can be managed.

Fusarium spp. Contamination of Wheat, Maize, Soybean, and Pea Grain in Croatia

Fusarium spp. Contamination of Wheat, Maize, Soybean, and Pea Grain in Croatia From 2002 to 2008, 203 samples of wheat, maize, soybean, and pea were analysed for the presence of Fusarium species. Contamination with Fusarium spp., expressed as the percentage of seeds with Fusarium colonies, ranged from 5 % to 69 % for wheat, from 25 % to 100 % for maize, from 4 % to 17 % for soybean, and from 3 % to 17 % for pea. 187 isolates were collected and the following 19 species determined: F. graminearum, F. poae, F. avenaceum, F. verticillioides, F. sporotrichioides, F. heterosporum, F. crookwellense, F. tricinctum, F. semitectum, F. oxysporum, F. proliferatum, F. solani, F. equiseti, F. pseudograminearum, F. chlamydosporum, F. sambucinum, F. compactum, F. scirpi, and F. culmorum. Dominant species were F. graminearum on wheat (27 % of isolates), F. verticillioides on maize (83 % of isolates), F. sporotrichioides on soybean (34 % of isolates), and F. proliferatum on pea (29 % of isolates). Among species identified, F. heterosporum, F. crookwellense, F. pseudograminearum, F. sambucinum, and F. compactum have been reported for the first time in Croatia. Kontaminacija zrna pšenice, kukuruza, soje i graška vrstama fusariuma u Hrvatskoj U periodu od 2002. do 2008. g. analizirana je prisutnost vrsta Fusariuma na 208 uzoraka zrna pšenice, kukuruza, soje i graška. Kontaminacija vrstama Fusariuma, izražena kao postotak sjemenki s kolonijama Fusarium spp., kretala se od 5 % do 69 % na pšenici, od 25 % do 100 % na kukuruzu, od 4 % do 17 % na soji te od 3 % do 17 % na grašku. Prosječna kontaminacija vrstama Fusariuma u različitim godinama varirala je od 10 % do 46 % na pšenici, od 50 % do 91 % na kukuruzu, od 5 % do 9 % na soji te od 7 % do 10 % na grašku. Vrste Fusariuma koje se javljaju na zrnu izolirane su i determinirane s odabranih uzoraka pšenice, kukuruza, soje i graška. Skupljeno je 187 izolata, a utvrđeno je 19 vrsta: F. graminearum, F. poae, F. avenaceum, F. verticillioides, F. sporotrichioides, F. heterosporum, F. crookwellense, F. tricinctum, F. semitectum, F. oxysporum, F. proliferatum, F. solani, F. equiseti, F. pseudograminearum, F. chlamydosporum, F. sambucinum, F. compactum, F. scirpi i F. culmorum. Dominantne vrste bile su F. graminearum na pšenici (27 % izolata), F. verticillioides na kukuruzu (83 % izolata), F. sporotrichioides na soji (34 % izolata) te F. proliferatum na grašku (29 % izolata). U Hrvatskoj su prvi put utvrđene vrste F. heterosporum, F. crookwellense, F. pseudograminearum, F. sambucinum i F. compactum.

In vitro sensitivity of Fusarium graminearum, F. avenaceum and F. verticillioides to carbendazim, tebuconazole, flutriafol, metconazole and prochloraz

Growth of 13 F. graminearum isolates, 6 F. avenaceum isolates and 6 F. verticillioides isolates was analysed on potato-dextrose agar amended with 0.1, 0.33, 1, 3.3 and 10 mg l-1 of carbendazim, tebuconazole, flutriafol, metconazole, and prochloraz. Average concentration which reduced mycelial growth by 50% comparing it to control (EC50) was calculated for each isolate. Among fungicides tested, prochloraz was shown to be the most effective in growth inhibition of all three species, while flutirafol was proven to be the least effective. Metocnazole was more efficient in comparison with carbendazim and tebuconazole. EC50 values of all isolates on prochloraz were lower than 0.1 mg l-1, while on flutirafol they ranged between 1.66 and 8.51 mg l-1 for 18 isolates, or were higher than 10 mg l-1 for 7 isolates. EC50 values on carbendazim were 0.39-1.41 mg l-1 for F. graminearum isolates, 0.91-1.35 mg l-1 for F. avenaceum, and 0.47-0.6 mg l-1 for F. verticillioides. EC50 values on tebuconazole were 0.85- 2.57 mg l-1 for F. graminearum, 0.85-1.58 mg l-1 for F. avenaceum and 0.22-0.85 mg l-1 for F. verticillioides, while on metconazole EC50 values ranged between less than 0.1 mg l-1 to 1.66, 0.56, and 0.17 mg l-1 for F. graminearum, F. avenaceum and F. verticillioides, respectively. Average growth inhibitions of different Fusarium species and all Fusarium isolates together on different concentrations of fungicides tested were significantly different. Significant differences in growth were not determined among isolates of the same species on neither one of fungicides tested, indicating that no decreased sensitivity to the fungicides exists among isolates included in the study.

Identification of Fusarium Species Isolated from Stored Apple Fruit in Croatia

Abstract Several species of the genus Fusarium can cause apple fruit to rot while stored. Since Fusarium taxonomy is very complex and has constantly been revised and updated over the last years, the aim of this study was to identify Fusarium species from rotten apples, based on combined morphological characteristics and molecular data. We identified 32 Fusarium isolates from rotten apple fruit of cultivars Golden Delicious, Jonagold, Idared, and Pink Lady, stored in Ultra Low Oxygen (ULO) conditions. Fusarium rot was detected in 9.4 % to 33.2 % of naturally infected apples, depending on the cultivar. The symptoms were similar in all four cultivars: a soft circular brown necrosis of different extent, with or without visible sporulation. Fusarium species were identified by the morphology of cultures grown on potato-dextrose agar (PDA) and carnation leaf agar (CLA). Twenty one isolates were identified as Fusarium avenaceum and confirmed as such with polymerase chain reaction (PCR) using specific primer pair FA-ITSF and FA-ITSR. F. pseudograminearum,F. semitectum, F. crookwellense, and F. compactum were identified by morphological characteristics. F.avenaceum can produce several mycotoxins and its dominance in Fusarium rot points to the risk of mycotoxin contamination of apple fruit juices and other products for human consumption. Pathogenicity tests showed typical symptoms of Fusarium rot in most of the inoculated wounded apple fruits. In this respect Fusarium avenaceum, as the dominant cause of Fusarium rot in stored apple fruits is a typical wound parasite. Fuzarijsku trulež ploda jabuke može uzrokovati veći broj vrsta roda Fusarium. Budući da je taksonomija roda Fusarium vrlo kompleksna te je podložna neprestanim promjenama posljednjih godina, cilj ovog rada bio je identificirati vrste roda Fusarium, izolirane sa zaraženih plodova jabuke na temelju morfoloških karakteristika i molekularnih analiza. Skupljena su ukupno 32 izolata roda Fusarium sa zaraženih plodova jabuke kultivara Golden Delicious, Jonagold, Idared i Pink Lady, nakon skladištenja u ULO („Ultra Low Oxygen“) uvjetima. Fuzarijska je trulež zabilježena na 9,4 % do 33,2 % plodova zahvaćenih skladišnim bolestima. Simptomi fuzarijske truleži bili su slični kod sva četiri kultivara, a javljali su se u obliku smeđe, mekane nekroze koja se postepeno širi, uz pojavu ili bez pojave vidljive sporulacije na površini ploda. Vrste su identificirane na temelju morfoloških karakteristika na krumpirsko-dekstroznom agaru (KDA) i agaru s listićem karanfila (LKA). Ukupno je 21 izolat determiniran kao vrsta Fusarium avenaceum, što je potvrđeno i lančanom reakcijom polimerazom (PCR) uz uporabu specifičnog para početnica FA-ITSF i FA-ITSR. Vrste F.pseudograminearum, F. semitectum, F. crookwellense i F. compactum identificirane su na temelju morfologije. Istraživanjem je utvrđeno da je F. avenaceum, vrsta koja je potencijalni proizvođač nekoliko mikotoksina, dominantan uzročnik fuzarijske truleži ploda jabuke nakon skladištenja. Visok postotak zaraze fuzarijskom truleži može izazvati kontaminaciju sokova jabuke i ostalih proizvoda mikotoksinima, sekundarnim metabolitima vrsta Fusarium štetnih za ljudsko zdravlje. Testovi patogenosti pokazali su da većina izolata uzrokuje pojavu tipičnih simptoma fuzarijske truleži nakon inokulacije na oštećeni plod. Navedeno upućuje na činjenicu da je F. avenaceum, kao dominantan uzročnik fuzarijske truleži ploda jabuke tipičan parazit rana

The first report on mushroom green mould disease in Croatia

Abstract Green mould disease, caused by Trichoderma species, is a severe problem for mushroom growers worldwide, including Croatia. Trichoderma strains were isolated from green mould-affected Agaricus bisporus (button or common mushroom) compost and Pleurotus ostreatus (oyster mushroom) substrate samples collected from Croatian mushroom farms. The causal agents of green mould disease in the oyster mushroom were T. pleurotum and T. pleuroticola, similar to other countries. At the same time, the pathogen of A. bisporus was exclusively the species T. harzianum, which is different from earlier findings and indicates that the range of mushroom pathogens is widening. The temperature profiles of the isolates and their hosts overlapped, thus no range was found that would allow optimal growth of the mushrooms without mould contamination. Ferulic acid and certain phenolic compounds, such as thymol showed remarkable fungistatic effect on the Trichoderma isolates, but inhibited the host mushrooms as well. However, commercial fungicides prochloraz and carbendazim were effective agents for pest management. This is the first report on green mould disease of cultivated mushrooms in Croatia Bolest zelene plijesni uzrokovane vrstama roda Trichoderma velik je problem pri uzgoju gljiva u cijelom svijetu, uključujući i Hrvatsku. Vrste Trichoderma izolirane su iz komposta onečišćenog zelenom plijesni pri uzgoju šampinjona (Agaricus bisporus), kao i iz uzoraka supstrata uzgoja bukovača (Pleurotus ostreatus), s farma gljiva u Hrvatskoj. Pri infekciji bukovača izolirani su i identificirani uzročnici vrsta Trichodermapleurotum i T. pleuroticola, što odgovara nalazima u drugim zemljama, dok je iz uzgoja šampinjona izolirana samo vrsta T. harzianum. Navedeni su podaci različiti od prijašnjih nalaza i upućuju na to da se širi broj infektivnih uzročnika pri uzgoju gljiva. Temperaturni profil izolata i njihovih domaćina preklapao se, a komercijalni fungicidi prokloraz i karbendazim nađeni su kao potencijalno dobri kandidati za učinkovito suzbijanje ovih infekcija. Ferulična kiselina i neke fenolne tvari kao što je timol pokazuju značajan fungistatski učinak na izolate vrsta roda Trichoderma, ali su također inhibitorni i za domaćine - gljive. Ovo je prvo izvješće o bolesti izazvanoj zelenom plijesni pri uzgoju gljiva šampinjona i bukovača u Hrvatskoj.

Effects of Ocimum spp. essential oil on Monilinia laxa in vitro

One of the most important postharvest diseases of peach and nectarine is brown rot caused by the fungus Monilinia laxa. Increasing concern related to the toxic effect of the pesticide residues on fruits has enhanced the interest for the control of postharvest pathogenic fungi using biologically active constituents of plants such as essential oils. Species from genus Ocimum are known to possess essential oils with antifungal and fungicidal properties. The present paper reports the results of essential oil composition of four basil taxa and preliminary screening of their antifungal activity against M. laxa. The greatest effectiveness was achieved by the essential oils from Ocimum basilicum var. purpurascens and Ocimum tenuiflorum, whose activity was high throughout the testing period of twenty-three days.

Pathogenicity and potential toxigenicity of seed-borne Fusarium species on soybean and pea.

The effect of 15 Fusarium species on seed germination and early plant development was examined in a laboratory study, together with the potential toxigenicity of selected isolates and the possibility of T-2 toxin production on soybean and pea grain. In germination tests, 33 out of 47 isolates of Fusarium chlamydosporum, F. pseudograminearum, F. sporotrichioides, F. crookwellense, F. verticillioides, F. equiseti, F. semitectum, F. solani, F. poae, F. sambucinum and F. compactum significantly reduced the number of normal soybean seedlings, while only six out of 48 isolates of F. sporotrichioides, F. semitectum and F. chlamydosporum significantly reduced the number of normal pea seedlings. When inoculated on plants grown in Hoaglands media, nearly all Fusarium isolates caused necrosis of soybean and pea root, but neither of them significantly reduced shoot and root dry mass of pea plants, or shoot dry mass of soybean plants. Amplification of tri5 and FUM1 genes, required for trichothecene and fumonisin biosynthesis, was carried out by PCR in 45 Fusarium isolates from soybean and 40 isolates from pea. Positive tri5 PCR reaction was recorded in 19 isolates of F. sporotrichioides, F. crookwellense, F. pseudograminearum, F. poae, F. sambucinum, F. culmorum, F. equiseti and F. chlamydosporum. Positive FUM1 PCR reaction was recorded in 35 out of 38 F. verticillioides and F. proliferatum isolates. The content of T-2 toxin produced by eight F. sporotrichioides isolates inoculated on autoclaved soybean, pea and barley grain ranged from 69.4 to 2595.5 μg/kg. No significant differences were determined between T-2 toxin production on soybean and barley grain, nor on pea and barley grain.

Identification of Fusarium Species Isolated From Stored Apple Fruit in Croatia / Identifikacija Vrsta Roda Fusarium Izoliranih S Plodova Jabuke Nakon Skladištenja

Abstract Several species of the genus Fusarium can cause apple fruit to rot while stored. Since Fusarium taxonomy is very complex and has constantly been revised and updated over the last years, the aim of this study was to identify Fusarium species from rotten apples, based on combined morphological characteristics and molecular data. We identified 32 Fusarium isolates from rotten apple fruit of cultivars Golden Delicious, Jonagold, Idared, and Pink Lady, stored in Ultra Low Oxygen (ULO) conditions. Fusarium rot was detected in 9.4 % to 33.2 % of naturally infected apples, depending on the cultivar. The symptoms were similar in all four cultivars: a soft circular brown necrosis of different extent, with or without visible sporulation. Fusarium species were identified by the morphology of cultures grown on potato-dextrose agar (PDA) and carnation leaf agar (CLA). Twenty one isolates were identified as Fusarium avenaceum and confirmed as such with polymerase chain reaction (PCR) using specific primer pair FA-ITSF and FA-ITSR. F. pseudograminearum,F. semitectum, F. crookwellense, and F. compactum were identified by morphological characteristics. F.avenaceum can produce several mycotoxins and its dominance in Fusarium rot points to the risk of mycotoxin contamination of apple fruit juices and other products for human consumption. Pathogenicity tests showed typical symptoms of Fusarium rot in most of the inoculated wounded apple fruits. In this respect Fusarium avenaceum, as the dominant cause of Fusarium rot in stored apple fruits is a typical wound parasite. Fuzarijsku trulež ploda jabuke može uzrokovati veći broj vrsta roda Fusarium. Budući da je taksonomija roda Fusarium vrlo kompleksna te je podložna neprestanim promjenama posljednjih godina, cilj ovog rada bio je identificirati vrste roda Fusarium, izolirane sa zaraženih plodova jabuke na temelju morfoloških karakteristika i molekularnih analiza. Skupljena su ukupno 32 izolata roda Fusarium sa zaraženih plodova jabuke kultivara Golden Delicious, Jonagold, Idared i Pink Lady, nakon skladištenja u ULO („Ultra Low Oxygen“) uvjetima. Fuzarijska je trulež zabilježena na 9,4 % do 33,2 % plodova zahvaćenih skladišnim bolestima. Simptomi fuzarijske truleži bili su slični kod sva četiri kultivara, a javljali su se u obliku smeđe, mekane nekroze koja se postepeno širi, uz pojavu ili bez pojave vidljive sporulacije na površini ploda. Vrste su identificirane na temelju morfoloških karakteristika na krumpirsko-dekstroznom agaru (KDA) i agaru s listićem karanfila (LKA). Ukupno je 21 izolat determiniran kao vrsta Fusarium avenaceum, što je potvrđeno i lančanom reakcijom polimerazom (PCR) uz uporabu specifičnog para početnica FA-ITSF i FA-ITSR. Vrste F.pseudograminearum, F. semitectum, F. crookwellense i F. compactum identificirane su na temelju morfologije. Istraživanjem je utvrđeno da je F. avenaceum, vrsta koja je potencijalni proizvođač nekoliko mikotoksina, dominantan uzročnik fuzarijske truleži ploda jabuke nakon skladištenja. Visok postotak zaraze fuzarijskom truleži može izazvati kontaminaciju sokova jabuke i ostalih proizvoda mikotoksinima, sekundarnim metabolitima vrsta Fusarium štetnih za ljudsko zdravlje. Testovi patogenosti pokazali su da većina izolata uzrokuje pojavu tipičnih simptoma fuzarijske truleži nakon inokulacije na oštećeni plod. Navedeno upućuje na činjenicu da je F. avenaceum, kao dominantan uzročnik fuzarijske truleži ploda jabuke tipičan parazit rana

Identifikacija vrsta roda Fusarium izoliranih s plodova jabuke nakon skladištenja

Abstract Several species of the genus Fusarium can cause apple fruit to rot while stored. Since Fusarium taxonomy is very complex and has constantly been revised and updated over the last years, the aim of this study was to identify Fusarium species from rotten apples, based on combined morphological characteristics and molecular data. We identified 32 Fusarium isolates from rotten apple fruit of cultivars Golden Delicious, Jonagold, Idared, and Pink Lady, stored in Ultra Low Oxygen (ULO) conditions. Fusarium rot was detected in 9.4 % to 33.2 % of naturally infected apples, depending on the cultivar. The symptoms were similar in all four cultivars: a soft circular brown necrosis of different extent, with or without visible sporulation. Fusarium species were identified by the morphology of cultures grown on potato-dextrose agar (PDA) and carnation leaf agar (CLA). Twenty one isolates were identified as Fusarium avenaceum and confirmed as such with polymerase chain reaction (PCR) using specific primer pair FA-ITSF and FA-ITSR. F. pseudograminearum,F. semitectum, F. crookwellense, and F. compactum were identified by morphological characteristics. F.avenaceum can produce several mycotoxins and its dominance in Fusarium rot points to the risk of mycotoxin contamination of apple fruit juices and other products for human consumption. Pathogenicity tests showed typical symptoms of Fusarium rot in most of the inoculated wounded apple fruits. In this respect Fusarium avenaceum, as the dominant cause of Fusarium rot in stored apple fruits is a typical wound parasite. Fuzarijsku trulež ploda jabuke može uzrokovati veći broj vrsta roda Fusarium. Budući da je taksonomija roda Fusarium vrlo kompleksna te je podložna neprestanim promjenama posljednjih godina, cilj ovog rada bio je identificirati vrste roda Fusarium, izolirane sa zaraženih plodova jabuke na temelju morfoloških karakteristika i molekularnih analiza. Skupljena su ukupno 32 izolata roda Fusarium sa zaraženih plodova jabuke kultivara Golden Delicious, Jonagold, Idared i Pink Lady, nakon skladištenja u ULO („Ultra Low Oxygen“) uvjetima. Fuzarijska je trulež zabilježena na 9,4 % do 33,2 % plodova zahvaćenih skladišnim bolestima. Simptomi fuzarijske truleži bili su slični kod sva četiri kultivara, a javljali su se u obliku smeđe, mekane nekroze koja se postepeno širi, uz pojavu ili bez pojave vidljive sporulacije na površini ploda. Vrste su identificirane na temelju morfoloških karakteristika na krumpirsko-dekstroznom agaru (KDA) i agaru s listićem karanfila (LKA). Ukupno je 21 izolat determiniran kao vrsta Fusarium avenaceum, što je potvrđeno i lančanom reakcijom polimerazom (PCR) uz uporabu specifičnog para početnica FA-ITSF i FA-ITSR. Vrste F.pseudograminearum, F. semitectum, F. crookwellense i F. compactum identificirane su na temelju morfologije. Istraživanjem je utvrđeno da je F. avenaceum, vrsta koja je potencijalni proizvođač nekoliko mikotoksina, dominantan uzročnik fuzarijske truleži ploda jabuke nakon skladištenja. Visok postotak zaraze fuzarijskom truleži može izazvati kontaminaciju sokova jabuke i ostalih proizvoda mikotoksinima, sekundarnim metabolitima vrsta Fusarium štetnih za ljudsko zdravlje. Testovi patogenosti pokazali su da većina izolata uzrokuje pojavu tipičnih simptoma fuzarijske truleži nakon inokulacije na oštećeni plod. Navedeno upućuje na činjenicu da je F. avenaceum, kao dominantan uzročnik fuzarijske truleži ploda jabuke tipičan parazit rana