Ferenc Bagi

The protective effect of hulls on the occurrence of Alternaria mycotoxins in spelt wheat

BACKGROUND Since there is an increasing demand on the world market for alternative crops suitable for organic production, spelt wheat (Triticum aestivum spp. spelta L.) is a highly attractive farming option. Alternaria species are widespread and infect a great variety of economically important crops. Certain species are known producers of mycotoxins. The aim of this study was to assess the protective effect of hulls covering the spelt kernels on Alternaria toxins. RESULTS Alternariol (AOH) and alternariol monomethyl ether (AME) were evaluated in hulls and dehulled kernels after plant inoculation with one A. alternata and two different A. tenuissima isolates. Mycotoxins were determinated using high-performance liquid chromatography with dioade array detection. The detected levels of AOH and AME were four times higher in hulls compared to kernels in inoculation treatments. AOH was registered at levels ranging from 227 to 331 µg kg(-1) in dehulled kernels and from 433 up to 1647 µg kg(-1) in hulls. AME was predominant toxin detected in the range of 277 to 398 µg kg(-1) in dehulled kernels and from 1844 to 2183 µg kg(-1) in hulls, with highly significant difference to water control treatment. CONCLUSION Obtained results indicate the significantly higher concentrations of Alternaria toxins in hulls than in dehulled kernels which implicate the possible protective effect of spelt wheat hulls.

Application of liquid chromatography with diode-array detector for determination of acetamiprid and 6-chloronicotinic acid residues in sweet cherry samples.

SUMMARY A rapid and simple method for simultaneous determination of acetamiprid and its metabolite 6-chloronicotinic acid in sweet cherry samples has been developed. This residue analysis method is based on the reversed phase separation on C18 column with gradient elution. Analytes’ determination and quantification were performed by high performance liquid chromatography (HPLC) with diode-array detector and chromatograms were extracted at 230 nm. Extraction efficiency experiments demonstrated the ability of this method to extract neonicotinoids from sweet cherry samples. These insecticides were extracted with a mixture of acetonitril/0.1N ammonium-chloride (8/2, v/v). The average recoveries of acetamiprid and 6-chlornicotinic acid from sweet cherry samples were in the range of 95-101% and 73-83%, respectively, with the associated relative standard deviations (RSDs) <5%. Expanded measurement uncertainties for the analyzed compounds were 2.7 and 3.01%. The limit of quantification (LOQ) was 10 µg/kg and 30 µg/kg for acetamiprid and 6-chloronicotinic acid, respectively. Thus, the developed HPLC/DAD method can be considered a useful tool for sensitive and rapid determination of acetamiprid and 6-chloronicotinic acid. Hence, the method may find further application in the analysis of real sweet cherry samples contaminated with these insecticides at a ppb level.

Analysis of Rhizoctonia solani isolates associated with sugar beet crown and root rot from Serbia

Rhizoctonia solani is one of the most important sugar beet pathogens worldwide with anastomosis groups (AGs) 2-2 and 4 as the most pathogenic strains on sugar beet. AG 2-2 (intraspecific groups IIIB and IV) can cause both root and crown rot and damping-off, while AG-4 is typically associated only with seedlings damping-off. A total of 20 isolates of Rhizoctonia spp. from sugar beet roots, showing characteristic crown and root rot symptoms, were collected from 4 localities in Serbia. Regarding colony morphology and cultural characteristics, they were divided into 2 groups, which corresponded to their pathogenic, anastomosis and molecular traits. Sequence analysis proved that the first group of isolates were closely related (sequence homology 100%) to AG-4, subgroup HG II, whereas the second group was determined to belong to AG 2-2 IIIB (sequence homology 99%). These two groups differed in range of hosts and in disease intensity on sugar beet, bean and soybean plants. This is the first detailed report on R. solani anastomosis groups that cause sugar beet crown and root rot in Serbia.

Susceptibility level of cucumber downy mildew (Pseudoperonospora cubensis) to metalaxyl

Level of susceptibility of Pseudoperonospora cubensis isolate from Ratkovo to metalaxyl in concentrations 50, 100, 200, 400 and 800 μg/ml was investigated. The trials were conducted on cotyledon and fully developed young leaves using cucumber cultivar Haros. Reduced level of susceptibility was detected in metalaxyl concentrations of 50, 100 and 200 μg/ml because the intensity of sporulation in these treatments was on the same level as in control. Sporulation was also observed on developed leaves treated with metalaxyl in concentrations of 400 and 800 μg/ml.

Evaluation of rapid protocols for DNA isolation from Cercospora beticola Sacc.

Summary: The most fungal DNA isolation protocols are designed to obtain high amounts of very pure DNA, requiring large fungal cultures and extraction procedures with many purification steps. Since the PCR does not require high purity DNA, the aim of this investigation was to evaluate three fast and simple fungal DNA isolation protocols for further use in Cercospora PCR based research. The purity and quantity of isolated DNAs were determined spectrophotometrically, electrophoretically and by PCR reaction with universal primers. The amounts of DNA evaluated on agarose gels, isolated by protocols A and C, did not correspond to the spectrophotometrical values, probably due to RNA impurities. In samples isolated by protocol B these impurities were not detected and the DNA concentrations were more similar. Neither protocol eliminated impurities such as carbohydrates and phenol. The average DNA yield of protocol A was 1.04 µg/µl, protocol B 0.88 µg/µl, and protocol C 0.55 µg/µl. The DNA quality most suitable for PCR analysis was obtained by protocol A, where amplification product with universal primers was detected in all DNA samples. The amplification product was detected in 87% of samples isolated by protocol C and in only 60% of samples isolated by protocol B. Although DNA obtained by protocol A had the highest yield and best quality, the isolation protocol C should be also recommended, for it does not require phenol, chlorophorm or liquid nitrogen.Key words: Cercospora, DNA, impurity, isolation, protocol

THE EFFECT OF FUNGICIDE TREATMENT ON MYCOTOXIN CONTENT AND YIELD PARAMETERS OF WHEAT

Effects of treatment with triazole fungicide were evaluated on 14 wheat genotypes with respect to mycotoxin (aflatoxins, ochratoxin A and deoxynivalenol), yield, 1000 kernel weight and hectoliter weight. Mycopopulation of seed samples was also determined. According to the results, fungicide treatment can reduce the level of mycotoxins in seed samples in order to improve the quality parameters and reduce the level of fungal contamination.

PATHOGENIC, MORPHOLOGICAL AND MOLECULAR CHARACTERISTICS OF ALTERNARIA TENUISSIMA FROM SOYBEAN

During 2008 and 2009 phytopathological isolations were done from soybean plants and seed samples from several localities in Serbia. A total of 19 isolates of Alternaria spp. were isolated, 13 from the seed and 3 from both leaf and stem. In order to determine and characterize isolates, cultural, morphological, molecular and pathogenic characteristics were thoroughly investigated. The slowest growth of the examined isolates was noted on Malt agar (MA) with average colony diameter of 42.9 mm after 7 days of incubation. On other two media (V8 and PCA), colony growth was uniform and faster, with average diameter of 66.8 mm and 66.1 mm, respectively. Isolates of fungi form unbranched or poorly branched conidial chains on short unbranched conidiophores. Conidia are dark in colour, multicellular with transverse and longitudinal septae. They are of different size regarding the place of formation in the chain. Based on these characteristics, the tested isolates were determined as Alternaria tenuissima. Molecular identification with sequencing of ITS1-5.8S-ITS2 rDNA verified that investigated isolates belong to Alternaria tenuissima group. Pathogenicity test proved that all isolates were more or less virulent to soybean seed (12.5% to 40% of rotten seeds), while pathogenicity on plants was poorly expressed.

A New Concept to Secure Food Safety Standards against Fusarium Species and Aspergillus Flavus and Their Toxins in Maize

Commercial maize hybrids are exposed to different degrees of ear infection by toxigenic fungal species and toxin contamination. Their resistance to different fungi and toxin relationships are largely unknown. Without this knowledge, screening and breeding are not possible for these pathogens. Seven- to tenfold differences were found in resistance to Fusarium spp., and there was a five-fold difference in ear coverage (%) in response to A. flavus. Three hybrids of the twenty entries had lower infection severity compared with the general means for toxigenic species. Three were highly susceptible to each, and 14 hybrids reacted differently to the different fungi. Differences were also observed in the toxin content. Again, three hybrids had lower toxin content in response to all toxigenic species, one had higher values for all, and 16 had variable resistance levels. Correlations between infection severity and deoxynivalenol (DON) content were 0.95 and 0.82 (p = 0.001) for F. graminearum and F. culmorum, respectively. For fumonisin and F. verticillioides ear rot, the Pearson correlation coefficient (r) was 0.45 (p = 0.05). Two independent isolates with different aggressiveness were used, and their mean X values better described the resistance levels. This increased the reliability of the data. With the introduction of this methodological concept (testing the resistance levels separately for different fungi and with two isolates independently), highly significant resistance differences were found. The resistance to different fungal species correlated only in certain cases; thus, each should be tested separately. This is very useful in registration tests and post-registration screening and breeding. This would allow a rapid increase in food and feed safety.

Aflatoxin, zearalenone, deoxynivalenol and fumonisin contamination of maize from the Autonomous Province of Vojvodina

Aflatoxins (AFs), fumonisins (FUMs), zearalenone (ZEA) and deoxynivalenol (DON) have been recognized as major contaminants of maize. Therefore, the presence of AFs, ZEA, DON and FUMs was examined in a total of 100 maize samples from the Autonomous Province of Vojvodina. Sample analyses were performed using the Enzyme Linked Immunisorbent Assay method. The results obtained indicate that 74 % of the maize samples were contaminated with FUMs (540.1-5076 μg/kg), followed by 52 % contaminated with DON (275.2-882.1 μg/kg), 15 % contaminated with ZEA (35.6-183.5 μg/kg) and 5 % contaminated with AFs (2.28-4.31 μg/kg). Although 78 % of the samples were contaminated with at least one mycotoxin, the concentration of FUMs exceed the maximum level (ML) proscribed by the Serbian regulations only in 4 % of the samples. Furthermore, the detected concentrations of AFs, ZEA and DON were in accordance with national regulations. This study is the first research of its kind on the presence of AFs, ZEA, DON and FUMs in maize harvested in the APV in 2016.

Molecular characterization of Pseudomonas syringae pvs. from different host plants by repetitive sequence-based PCR and multiplex-PCR@@@Įvairių augalų-šeimininkų Pseudomonas syringae pvs. molekulinis apibūdinimas taikant pasikartojančių sekų ir jungtinę PGR

Pseudomonas syringae pvs., isolated from different cultivars of sweet cherry grown in several locations in Serbia, were characterized and compared with strains collected earlier from sour and sweet cherry and oil pumpkin growing in this region, as well as with reference strains P. syringae pv. morsprunorum race 1 CFBP2119, P. s. pv. lachrymans 765R and P. s. pv. syringae H-1. By employing LOPAT and GATTa tests, isolates were identified as P. syringae pv. syringae and P. s. pv. morsprunorum race 1. Simultaneous detection of syringomycin synthesis (syrB) and syringomycin secretion (syrD) genes in multiplex-polymerase chain reaction (m-PCR) was used for P. syringae pv. syringae confirmation. All isolates detected as P. syringae pv. morsprunorum race 1 after biochemical characterization were positive for cfl gene amplification. Using a repetitive sequence-based PCR (rep-PCR) both syringae and morsprunorum race 1 pathovars were clustered separately, with 42% similarity. No significant differences between isolates in each pathovar were noted, although they were collected from different sweet cherry cultivars and varying localities. The most similar to the P. syringae pv. syringae isolates was strain T6 with 19% similarity, followed by strain Tk21 from oil pumpkin – 25%. The isolates of both pathovars were detected on the same location (Selenca, Serbia) and the same cultivar (‘Merchant’), albeit in two different years.