Vera Stojšin

Screening for Polyphenol Compounds and Antioxidant Capacity of Sweet Cherry Fruits Infected with Monilinia Laxa

Summary Monilinia laxa Aderh. and Ruhl. is the predominant causal agent of brown rot disease of stone fruit orchards, especially sweet cherries. The objective of this study was to identify reaction in response of nine genotypes cherry, with different pomological properties, against brown rot. These genotypes were harvested at commercial maturity from orchard in the Fruit Research Institute in Rimski Šančevi. The studied genotypes showed significant differences in terms of the occurrence of disease on fruits, both under artificial inoculation and infection in the field. Given the fact that sweet cherry fruits are prone to infection by a number of pathogens in the field, biochemical parameters were analysed on artificially inoculated fruits. Biochemical analysis of fruits determined significant differences in contents of total phenols, flavonoids and anthocyanins, as well as in antioxidant activity. It was genotype specificities and intensity of infection, as well as the interaction of the two that induced differences in the secondary biomolecules content and antioxidant activity. The majority of the genotypes examined showed high polyphenolics content, while under the infection, the content was significantly lower. Based on the results obtained, the secondary metabolites content can be used as one of the parameters for evaluating the resistance of sweet cherry genotypes to brown rot.

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

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.

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.

Correlation between lipid peroxidation and phenolics content in leaves and roots of sugar beet infected with Rhizoctonia solani

The correlation between intensity of lipid peroxidation and changes in antioxidant capacity of sugar beet plants (cv. ‘Drena’) infected with Rhizoctonia solani Kühn isolate (AG 2-2 IIIB group) was studied. Successful inoculation was confirmed by the presence of infection cushions in a cross section of leaf petioles. On the 7th day of the experiment, phenylalanine ammonia-lyase (PAL; EC. 4.3.1.5) activity was in negative correlation with intensified lipid peroxidation process in leaves of sugar beet plants (r= –0 .99). Also, in leaves and roots of inoculated sugar beet plants, total flavonoids content (35% and 20%, respectively) and 1,1-diphenyl-2-picrylhydrazyl (DPPH)-scavenging activity (80% and 55%, respectively) were significantly reduced. Necrotic processes resulting from R. solani infection of sugar beet plants was followed by induction of plant phenolics metabolism; however, antioxidant capacity of these plants was reduced.

Gibberella intermedia the pathogen of St. John's wort, coneflower and marshmallow in Serbia.

Gibberella intermedia (Kuhlmann) Samuels et al. (anamorf: Fusarium proliferatum /Matsushima/ Nirenberg) was isolated from seeds of St. Johns wort, marshmallow, and coneflower, as well as from roots and stalks of marshmallow and roots of coneflower. These plants had symptoms of leaf chlorosis, malformation, withering and plant dwarfing and were collected from several localities in Serbia during five-year investigations of mycopopulations of the mentioned plants. The morphological characteristics of the pathogen were described.