J. Lević

A European Database of Fusarium graminearum and F. culmorum Trichothecene Genotypes

Fusarium species, particularly Fusarium graminearum and F. culmorum, are the main cause of trichothecene type B contamination in cereals. Data on the distribution of Fusarium trichothecene genotypes in cereals in Europe are scattered in time and space. Furthermore, a common core set of related variables (sampling method, host cultivar, previous crop, etc.) that would allow more effective analysis of factors influencing the spatial and temporal population distribution, is lacking. Consequently, based on the available data, it is difficult to identify factors influencing chemotype distribution and spread at the European level. Here we describe the results of a collaborative integrated work which aims (1) to characterize the trichothecene genotypes of strains from three Fusarium species, collected over the period 2000–2013 and (2) to enhance the standardization of epidemiological data collection. Information on host plant, country of origin, sampling location, year of sampling and previous crop of 1147 F. graminearum, 479 F. culmorum, and 3 F. cortaderiae strains obtained from 17 European countries was compiled and a map of trichothecene type B genotype distribution was plotted for each species. All information on the strains was collected in a freely accessible and updatable database (www.catalogueeu.luxmcc.lu), which will serve as a starting point for epidemiological analysis of potential spatial and temporal trichothecene genotype shifts in Europe. The analysis of the currently available European dataset showed that in F. graminearum, the predominant genotype was 15-acetyldeoxynivalenol (15-ADON) (82.9%), followed by 3-acetyldeoxynivalenol (3-ADON) (13.6%), and nivalenol (NIV) (3.5%). In F. culmorum, the prevalent genotype was 3-ADON (59.9%), while the NIV genotype accounted for the remaining 40.1%. Both, geographical and temporal patterns of trichothecene genotypes distribution were identified.

Fusarium species: the occurrence and the importance in agriculture of Serbia.

Fusarium species have been isolated from over 100 plant species in Serbia. From the economic aspect, they have been and still are the most important for the production and storage of small grains and maize, and are exceptionally important for some other species. Total of 63 species, 35 varieties (var.) and 19 specialised forms (f. sp.) of basic species, particularly of F. oxysporum (4 var. and 12 f. sp.) and F. solani (7 var. and 3 f. sp.) were identified. Species F. langsethiae and F. thapsinum, recently identified, have been isolated from wheat and s o r g h u m seeds, respectively. F. graminearum is the most important pathogen for wheat, barely and maize, while F. poae is also important for wheat and barely. Furthermore, species of the section Liseola (F. verticillioides, F. subglutinans and F. proliferatum) are important for maize and sorghum. In recent years, species of the section Liseola have been increasingly occurring in wheat and barley. The June-October period in Serbia is the most critical period for quality maintenance of stored maize, as the abundance and frequency of fungi, particularly of toxigenic species of the genus Fusarium, are the greatest during that period. In general, there is a lack of data about fusarioses of industrial crops in Serbia. There are mere descriptions of specific cases in which the development of Fusarium species was mostly emphasised by agroecological conditions. The presence of recently determined Fusarium species in kernels of these plant species indicates their importance from the aspect of the yield reduction and grain quality debasement and the mycotoxin contamination. Root rot and plant wilt are characteristic symptoms of fusarioses for forage and vegetable crops, while pathological changes in fruits provoked by Fusarium species are less frequent. F. oxysporum and its specialised forms prevail in these plant species.

Ubiquity of toxigenic fungi and mycotoxins in animal feeds in Republic of Serbia

Climatic conditions and growing of grain on large areas in Republic of Serbia are suitable for development of numerous toxigenic species, such as Fusarium spp., Aspergillus spp. and Penicillium spp., and resulting from this is frequent incidence of animal feed contamination by their toxic products. In Republic of Serbia, the most frequent fungi determined in animal feed are those from Fusarium genus, as well as their mycotoxins. Of Fusarium species the most frequent one is F. graminearum, and of toxins zearalenon, in maize or wheat grains, which are the main raw material in production of animal feeds. Development of this fungi and bio-synthesis of zearalenon most often depend on period of rains in the third decade of May when wheat is in the blossoming phenostage or on ample precipitation and low temperatures at the end of summer or beginning of autumn during sensitive pheno-stage of maize growing. Aflatoxins are rarely isolated in Republic of Serbia, but there are conditions for their increased presence in imported feed components. Natural occurrence of ochratoxin A is more frequent than incidence of aflatoxins in climatic conditions of Republic of Serbia. Presence of mycotoxins produced by species of genera Fusarium, Aspergillus and Penicillium indicates potential risk of incidence of mycotoxicosis, especially in younger animal categories. For the purpose of protection of health of animals, realization of profit and high productivity in livestock production, it is necessary to explain factors which can cause animal intoxication, and to identify toxins and develop methodology for assessment of the potential toxicity of animal feed.

The presence of toxigenic Fusarium species and Fusariotoxins deoxynivalenol and zearalenone in winter wheat

The frequency of fungi and mycotoxin concentrations of deoxynivalenol (DON) and zearalenone (ZON) were studied in winter wheat grains harvested in 2009. The most frequently isolated species belonged to genera Alternaria (81.55%) and Fusarium (12%), followed by Rhizopus spp. (3.75%), Acremoniella spp. (1.15%) and other fungi (Acremonium spp., Arthrinium spp., Aspergillus spp., Bipolaris spp., Chaetomium spp., Nigrospora spp., Penicillium spp. and Ramichloridium spp.) isolated in less than 1%. The following species of the genus Fusarium were identified: F. graminearum (82.50%), F. sporotrichioides (5.42%), F. proliferatum (4.17%), F. subglutinans (4.17%), F. poae (1.66%), F. semitectum (1.25%), and F. verticillioides (0.83%). In 100% of wheat grain samples DON was detected (110-1200 μg kg -1 , average 490 μg kg -1 ), while ZON was detected in 10% of samples and in the lower average of 70 μg kg -1 with the limit values ranging from 60 to 80 μg kg -1 . Statistically significant positive correlations were established between the concentration of ZON with the frequency of F. graminearum (r = 0.63**) or with the frequency of Fusarium spp. (r = 0.58**). A negative insignificant correlation was determined between the DON level and the percentage of present Fusarium species.

Fumonisin B1 in maize, wheat and barley grain in Serbia.

A wide variety of commodities in the world have been analyzed for fumonisins contamination. However, they have mostly been reported in maize and maize-based foods and feeds. Just a few scientific researches were conducted to obtain results on natural contamination of wheat and barley with these mycotoxins. This survey was conducted to evaluate fumonisin B1 contamination in maize, wheat and barley grain in Serbia. A total of 203 maize, 180 wheat and 120 barley samples were obtained from different local warehouses between October 2007 and June 2009. Concentration of FB1 were analysed with the Enzyme-Linked Immunosorbent Assay (ELISA). Positive results were found in 70.7%, 60.6% and 34.1%, in the maize, wheat and barley samples, respectively. FB 1 concentration varied from 750 to 4900 µg kg -1 , and the mean levels recorded were: 1225.7 kg -1 in maize; 852.7 µg kg -1 in wheat; and 768.2 µg kg -1 in barley. The mycotoxin contamination of cereals was affected by factors such as origin resistance, drought- stress, and insect damage and differed between the years of investigation The results obtained in this survey revealed that FB1 is frequent contaminant of cereal grains in Serbia. Considering that these products are consumed in large amounts either directly or as components of foods and feeds, the levels of contamination reported herein indicate a potential threat to animal and public health.

The use of vegetative compatibility tests for identification of biodiversity of phytopathogenic fungi.

SUMMARY Visual assessment of phenotypes, performed when two strains of one fungal species are cultivated in a mixed culture on specific media, is known as vegetative or heterokaryotic compatibility or incompatibility test, which enables identification of fungal clones and their classification based on phylogenetic groups. Hyphae of strains that have identical alleles at all vic loci can anastomose into a form of a visible heterokaryon. Strains that divide compatible loci and can anastomose each other belong to a subpopulation termed the vegetative compatibility group (VCG), which is genetically distinguishable from other VCGs. Each VCG is specific regarding its host plant or related host groups and can, but does not have to be virulent on other hosts. Vegetative compatibility can be established in different ways, but complementary auxotrophic strains or strains formed by spontaneous mutation during nutrition, capable of forming a prototrophic heterokaryon are predominantly used. The nit mutants are considered excellent genetic markers for determination of vegetative compatibility and grouping of strains or clones of one fungus into the same or different VCGs. The ability only to determine whether strains are the same or not, but not the degree of their relatedness using VCG, is a limiting factor in analyses that could be performed. VCGs are the most efficient when they are employed to detect the presence of a specific strain in a population. This paper provides an overview of the importance of the phenomenon of vegetative compatibility. Vegetative compatibility is one of the most important genetic traits in ascomycetes by which one subpopulation can be identified as a distinct genetic group. Furthermore, the procedures for isolation, identification and determination of nit mutant phenotypes, and for identification of complementary strains and VCGs are described in detail.

Occurrence of Fusarium species in maize grains for silage.

Occurrence of Fusarium species in maize grains for silage Grain samples of two maize hybrids, medium early (ZP434) and late maturity (ZP704), collected during harvest in 2008 were investigated for contamination by fungi. Grains were plated on agar media and grown fungi were identified by morphological macroscopic and microscopic characteristics on potato-dextrose agar (PDA) and synthetic nutrient agar (SNA). Species of the genus Fusarium were the most common in both hybrids, and their presence amounted to 33.89% (ZP434) and 42.00% (ZP704). Other fungi of genera, Acremonium, Alternaria, Aspergillus, Chaetomium, Cladosporium, Nigrospora, Penicillium, Rhizopus and Trichotecium, were isolated from 0 to 41.00%. Four species belonging to the genus Fusarium were identified, of which the species F. verticillioides was the most common with 28.63% in ZP434 and 30.50% in ZP704 hybrids. The presence of F. graminearum, F. proliferatum and F. subglutinans ranged from 3.00% (ZP704) to 5.00% (ZP434), 0.13% (ZP434) to 7.00% (ZP704) and 0.13% (ZP434) to 7.00% (ZP704), respectively. Generally, the incidence of every particular fungus was higher in the late maize hybrid with a higher moisture content than in the medium-early hybrid with a lower moisture content.