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Leaf spot disease caused by Cercospora beticola Sacc. is the most destructive foliar disease of beet. Cercospora leaf spot is controlled primarily by fungicides because the non-chemical alternatives do not provide commercially viable control. One of the ways of reducing chemical application is the use of different essential oils (EOs) or antagonistic plant growth-promoting rhizobacteria (PGPB). This study evaluates several EOs and PGPB belonging to Pseudomonas chlororaphis as possible control agents of this pathogen. Antifungal properties were determined by in vitro microdilution method against five C. beticola monosporial isolates originated from the locality Brus, Serbia (53°53’ N, 21°04’E and 429 m above sea level) using EOs from medicinal plants: Turkish pickling herb (Echinophora tenuifolia), oregano (Origanum vulgare), basil (Ocimum basilicum), and myrtle (Myrtus communis) obtained by a hydro-distillation method. All tested oils displayed some antifungal activity against the fungal isolates. Origanum vulgare EO demonstrated the strongest antifungal activity (MIC - 0.0055±0.0051mg/mL), Ocimum basilicum slightly lower (MIC - 0.075±0.045mg/mL), followed by Myrtus communis (MIC - 0.775±0.045 mg/ mL) and Echinophora tenuifolia (MIC - 7.75±4.5 mg/mL). Five tested P. chlororaphis strains exhibited some antagonistic effect against C. beticola. Overnight culture (ONC) of P. chlororaphis strain E65 induced the highest percentage of inhibition (75.8%), followed by N3 (72.0%). A cell-free supernatant (CFS) and the CFS treated with EDTA (CFS-EDTA) of these strains showed similar inhibition of 60.2 and 56.0%, and both strains suppressed C. beticola growth. P. chlororaphis strains M1 and K113 also reduced the fungal growth by 67-70% using ONC and between 48-57% using different CFS fractions. The strains L1 and B25 caused inhibition of 60% using ONC and 50% by CFS. The lowest inhibition (~40%) by CFS-EDTA and heat-treated cell-free supernatant (HT-CFS) was recorded for B25, which was used as a reference strain. The tested isolates of C. beticola were susceptible to all selected essential oils and P. chlororaphis strains E25, N3, M1, and K113 in vitro, making them a promising non-chemical control agent. It is recommended that these findings should be tested in field conditions.

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.

Marshmallow is a host of a number of saprophytic and parasitic fungi in Serbia. The seeds of marshmallow are contaminated with fungi from different genera, especially Alternaria and Fusarium, which significantly reduced seed germination and caused seedling decay. In this study we investigate antagnonism of indigenous Bacillus sp. isolate Q3 against marshmallow mycopopulation. Bacillus sp. Q3 was isolated from maize rhizosphere, characterized by polyphasic approch and tested for plant growth promoting treats. Bacillus sp. Q3 produced antifungal metabolites with growth inhibition activity against numerous fungi in dual culture: 61.8% of Alternaria alternata, 74.8% of Myrothecium verrucaria and 33.6% of Sclerotinia sclerotiorum. That effect could be caused by different antifungal metabolites including siderophores, hydrolytic enzymes, organic acids and indole acetic acid (IAA). Suppression of natural marshmallow seed infection by Q3 isolate was observed. The seeds were immersed in different concentrations of bacterial suspension during 2h and their infections by phytopathogenic fungi were estimated. The results showed significant reduction of seed infection by Alternaria spp. The presented results indicate possible application of this isolate as promising biological agent for control of marshmallow seed pathogenic fungi.

Mycopopulation of yellow gentian growing in plantations was studied in 2008 and 2009. Fourteen species of fungi were registered at seed, out of which five were pathogenic. The most common species was Alternaria alternata (72-74 %). Species of the genus Fusarium (F. oxysporum, F. solani and F. equiseti) were present in a small percentage (2-6 %). These species, as well as F. verticillioides, were isolated from root, and Fusarium sp. was isolated from the flowers. Alternaria alternate, Epicoccum purpurescens, Phoma sp. and Alternaria sp. were regularly present on the leaves and stems.

Gibberella intermedia (Kuhlmann) Samuels et al. (anamorf: Fusarium proliferatum /Matsushima/ Nirenberg) was isolated from seeds of St. John's 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.

In preparation of samples for chromatographic determination of ochratoxin A, two types of columns were used for sample cleanup (SPE and immunoaffinity columns). The first method consisted of liquid-liquid extraction with a mixture of chloroform and phosphoric acid, followed by ion-exchange cleanup on Waters Oasis MAX columns. The sec­ond method consisted of extraction with a mixture of water and methanol, followed by LCTech OtaCLEAN immunoaf­finity column cleanup. Recoveries of the methods were determined at three levels in three repetitions for maize flour, and they were 84% (%RSD = 19.2) for the first method of sample preparation and 101% (%RSD = 2.2) for the second method. Values of LOQ for OTA were 0.25 and 1.00 μg/kg for the IAC and SPE clean-up procedures, respectively. Both methods comply with present regulations, but the MAX sample clean-up procedure should be used as an alternative, since the immunoaffinity column clean-up procedure is characterized by better reproducibility, accuracy, and efficiency.

Marshmallow is an important medicinal plant in Serbia. Because of increasing demands on the market, cultivation has been started. Through regular quality control of commercial seeds and plantations, mycopopulation of marshmallow was recorded in the period 2000-2006. Seeds of marshmallow were dominated by Alternaria alternata and species from the genus Fusarium (Fusarium verticillioides, F. proliferatum, F. semitectum, F. oxysporum and F. solani). Species belonging to Fusarium genus are the cause of rot of seeds and roots of marshmallow, causing chlorosis and fading, and therefore deterioration and necrosis of plants, as well as decrease of seed germination of seeds. Leaves and stalks of marshmallow were from time to time under massive attack of Puccinia malvacearum, and that was the reason why leaves were unuseful as a herbal drug. On roots and lower part of the stalks, massive appearance of Sclerotinia sclerotiorum, a causal agent of the white rot, in cases when marshmallow was cultivated after sunflower, was recorded, too. From other fungi in roots, species belonging to the genus Fusarium (F. oxysporum, F. solani and F. verticillioides) were dominant.

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. [ABSTRACT FROM AUTHOR]

Mycopopulation of yellow gentian growing in plantations was studied in 2008 and 2009. Fourteen species of fungi were registered at seed, out of which five were pathogenic. The most common species was Alternaria alternata (72-74 %). Species of the genus Fusarium (F. oxysporum, F. solani and F. equiset i) were present in a small percentage (2-6 %). These species, as well as F. verticillioides, were isolated from root, and Fusarium sp. was isolated from the flowers. Alternaria alternate, Epicoccum purpurescens, Phoma sp. and Alternaria sp. were regularly present on the leaves and stems. [ABSTRACT FROM AUTHOR]