BOTANICA SERBICA
Volume 40
Issue 1
2016

Editorial
Stevanovic, B.
Pages 1

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Professor Dr. Draga Simic, On the occasion of her 80th birthday
Kneževic-Vukcevic, J.
Pages 3-12

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Alfalfa (Medicago sativa L.) and Sinorhizobium meliloti:prospects of using rhizobial inoculants in Serbia
Delic, D., Stajkovic-Srbinovic, O., Kneževic-Vukcevic, J.
Pages 13-19

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ABSTRACT:  It is considered that the symbiotic association between alfalfa (Medicago sativa L.) and Sinorhizobium meliloti is one of the most efficient interactions between N₂-fixing bacteria and legumes. But even greater efficiency of symbiotic nitrogen fixation can be achieved by selecting the best combinations of host genotypes and nodule bacteria. The Serbian K-28 cultivar has the potential capacity to be a good parental genotype in breeding programs for improved N₂ fixation capability through inoculation with particular compatible strains. On the other hand, S. meliloti strain L5 is a broadly effective inoculant with most alfalfa cultivars, with a mean value of fixed N₂ of 35% over a twoyear period. Serbian soils are well supplied with autochthonous rhizobial strains which specifically nodulate alfalfa, and this is why the use of rhizobial inoculants in alfalfa production in Serbia is not a common practice. However, the number and effectiveness of such strains depend on soil type, the land use system (alfalfa stand, arable land and lea) and the alfalfa host plant. For this reason, use of rhizobial inoculants with highly effective strains is needed in alfalfa production. Our twoyear field experiment showed that shoot dry weight increased by 42-77% in inoculated alfalfa, while the amount of fixed nitrogen increased by 35%. Alfalfa inoculation with effective strains is an alternative approach to improving the long-term productivity of alfalfa. In our five-year trial, the inoculated K-28 alfalfa cultivar showed maximum nitrogen fixation (293 kg N ha^-1) in the fourth year of utilisation. During this period, symbiotic nitrogen fixation of alfalfa, determined from fixed N₂ in the second cutting, varied between 32 and 44%. The most effective strain promoted an increase of shoot dry weight by 34-60% in relation to the control in the third and fourth years of utilisation. In addition, Sinorhizobium spp. have the potential to be used as plant growth-promoting rhizobacteria (PGPR). Inoculation of Italian ryegrass seeds with a particular rhizobial strain the year before alfalfa growing ensured abundant nodulation and better growth of the alfalfa. In Serbia there are rhizobial inoculants of alfalfa with consistently high quality which permit reduction of mineral N fertiliser use in production of this crop, with consequent economic and ecological benefits.

KEY WORDS:  alfalfa, Sinorhizobium meliloti, symbiotic nitrogen fixation, rhizobial inoculants

Studying genotoxic and antimutagenic effects of plant extracts in Drosophila test systems
Stamenkovic-Radak, M., Andjelkovic M.
Pages 21-28

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ABSTRACT:  To gain more information on biological effects of plants, particularly herbs used in human medicine and diet, in vitro and in vivo methods have been developed to predict their genotoxicity and/ or antigenotoxicity in various test systems. The sex-linked recessive lethal (SLRL) and somatic mutations and recombination (SMART) tests are in vivo assays on D. melanogaster that have been used to test both mutagenic and antigenotoxic effects of extracts from numerous plant species used worldwide. The similarity of metabolic pathways between Drosophila and mammals and the ability to activate promutagens make the results of these tests widely applicable. Besides, Drosophila presents significant orthology with human genes that contr.ol cancers, which makes the assays on Drosophila reliable and informative for extrapolations onto humans.

KEY WORDS:  plant extracts, medicinal herbs, Drosophila, genotoxicity, antimutagenicity, diet

Optimisation of the microdilution method for detection of minimum inhibitory concentration values in selected bacteria
Kolarevic, S., Milovanovic, D., Avdovic, M., Oalde, M., Kostic, J., Sunjog, K., Nikolic, B., Kneževic-Vukcevic, J., Vukovic-Gacic, B.
Pages 29-36

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ABSTRACT:  In this study we investigated the influence of preparation of the bacterial inoculum for a microdilution susceptibility test, e.g., the effect of its optical density, on assessment of the minimum inhibitory concentrations (MIC). The approach employed in the majority of microdilution susceptibility studies is use of the same optical density for preparation of inoculums for different bacterial strains. In the present work, this approach was questioned by determining the ratio between the optical density and the number of bacteria in cultures. We also investigated whether the number of bacteria in inoculums can affect assessment of the MIC value for two antibiotics of broad spectra, rifampicin and streptomycin.
The study was performed on four Gram-positive and four Gram-negative bacteria (ATCC collection) commonly used to investigate antimicrobial potential. The ratio between the optical density and number of bacteria in cultures was determined for each strain, and a strong linear correlation was detected. However, it was evident that different bacteria have different cell numbers at the same OD₆₀₀ value. Based on the obtained results, inoculums for selected strains were prepared to obtain final cell numbers of 10³, 10⁴, 10⁵ and 10⁶ /well in the microdilution assay. Two different approaches were used in determining the MIC for rifampicin and streptomycin: approximation of MIC with IC90 and the resazurin reduction assay.
Our results indicated that the ratio between optical density and cell numbers is not constant and use of the same OD for inoculums for all strains can therefore lead to misinterpretation of the MIC values. We also observed influence of cell numbers in inoculums in determination of MIC values. For both approaches used (approximation of MIC with IC₉₀ and the resazurin reduction assay), the same trend was detected: antibiotics had the highest potency in experiments with the lowest bacteria cell number (10³/well). The lowest cell number (10³/well) is not recommended, as it can lead to false susceptibility results and to partial reduction of resazurin, which further complicates MIC determination. A final cell number of 10⁴/well can therefore be recommended as optimal.

KEY WORDS:  MIC, minimum inhibitory concentration, antibacterial activity, microdilution, resazurin

Antigenotoxic potential of plant monoterpenes linalool, myrcene and eucalyptol against IQ- and PhIP- induced DNA damage
Mitic-Culafic, D., Žegura, B., Filipic, M., Nikolic, B., Jovanovic, M., Kneževic-Vukcevic, J.
Pages 37-42

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ABSTRACT:  Nutrition is recognised as one of the major factors that can influence the development of cancer. Heterocyclic amines formed during the cooking of meat are genotoxic carcinogens and consumption of meat positively correlates with certain types of human cancer. On the other hand, it has been reported that many plant components reduce the genotoxic activities of carcinogens, including HCAs. In this study we investigated the antigenotoxic potential of the monoterpenes linalool, myrcene and eucalyptol against two food-borne carcinogens: 2-amino-1-methyl-6- phenylimidazo[4-5-b]pyridine (PhIP) and 2-amino-3-methylimidazo[4,5-f]-quinoline (IQ). The study was performed by the comet assay method in metabolically active HepG2 human hepatoma cells. In addition, the lipid peroxidation TBA assay was used to determine the anti-oxidative potential of these monoterpenes. To evaluate the antigenotoxic potential, cells were treated simultaneously with 0.01, 0.1 or 1 µg/mL of monoterpenes and either 90 µmol/LPhIP or 0.75 mmol/L IQ. The DNA damage induced by PhIP was reduced significantly (by 40-63%) by each of the three monoterpenes, while inhibition of IQ-induced DNA damage was less efficient (12-26% reduction) and limited to myrcene and eucalyptol. Lipid peroxidation was significantly inhibited by linalool and myrcene, with IC₅₀ values of approximately 30 µg/mL and 300 µg/mL, respectively. Eucalyptol showed only weak antioxidant activity (15% inhibition at 500 µg/mL). Taking into account the fact that reactive oxygen species (ROS) are formed during metabolic processing of PhIP, we assume that more efficient protection against PhIP-induced DNA damage was probably due to antioxidative activity of the monoterpenes or their metabolites, but the ability to interfere with metabolic activation of PhIP and IQ could also be involved.

KEY WORDS:  monoterpenes, heterocyclic amines, DNA damage, comet assay, HepG2 cells

Screening of the antibacterial effect of Juniperus sibirica and Juniperus sabina essential oils in a microtitre plate-based MIC assay
Nikolic, B., Vasiljevic, B., Mitic-Culafic, D., Lesjak, M., Vukovic-Gacic, B., Mimica Dukic, N., Kneževic-Vukcevic, J.
Pages 43-48

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ABSTRACT: The antibacterial effect of wild-growing Juniperus sibirica Burgsdorf and Juniperus sabina L. var. sabina essential oils was studied in a microtitre plate-based MIC assay. Bacterial growth was monitored by measuring turbidity of the sample (OD₆₀₀), as well as by following the colorimetric resazurin reaction. Essential oils were prepared from the needles of female plant samples and analysed by GC-MS. Hydrocarbon monoterpenes were determined as the dominant constituents; the compounds detected in the highest amounts were a-pinene (74.5%) and sabinene (54.3%) in J. sibirica and J. sabina oil, respectively. As indicator strains in the MIC assay, we used selected Grampositive (Enterococcus faecalis ATCC29212, Staphylococcus aureus ATCC25923, Bacillus subtilis ATCC6633 and Listeria innocua ATCC33090) and Gram-negative (Escherichia coli ATCC25922, Salmonella typhimurium ATCC14028, Salmonella enteritidis ATCC13076, Aeromonas hydrophila ATCC49140) bacteria. Bacterial inocula used in the MIC assay were adjusted to a 0.5 McFarland standard, corresponding to approximately 108 CFU/mL. The obtained results indicated that determination of turbidity decrease cannot be used to precisely quantify MIC values of the oils. The resazurin-incorporated MIC assay showed that the most susceptible strains were A. hydrophila and B. subtilis, with MIC values of 12.5 mg/mL and 6.25 mg/mL, respectively, for J. sibirica, and 6.25 mg/mL for both bacteria for J. sabina. The remaining bacteria were far less sensitive to Juniperus oils. In the range of tested concentrations, the effect of both oils was predominantly bactericidal, but J. sibirica oil showed a bacteriostatic effect against some Gram-negative bacteria.

KEY WORDS:  Juniperus sibirica, Juniperus sabina, essential oils, MIC assay

Determination of cyclodextrin production by cyclodextrin glycosyltransferase from alkalophilic Bacillus circulans strain B-65
Stankovic, S., Pešic, D., Beric, T., Simic, D.
Pages 49-54

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ABSTRACT:  New alkalophilic Bacillus sp. producers of cyclodextrin glycosyltransferase (CGTase) were isolated from 54 different soil samples (Serbian springs and soils). Amylolytic activity of isolates was found to be in the range of 0.4 to 12.2 U/mL and cyclodextrinogenic activity in the range of 0.02 to 0.53 U/ mL. The isolate designated as B-65 showed the highest amylolytic and cyclodextrinogenic activity. On the basis of morphological, physiological and biochemical characteristics, isolate B-65 was identified as an alkalophilic Bacillus circulans strain. Cyclodextrinogenic and amylolytic activity gradually increased over 96 h of incubation in a laboratory bioreactor. After 30 h of incubation, CGTase converted 40% of starch to cyclodextrins (CDs), whose final concentration reached 20 g/L. Analysis by HPLC revealed b-, a- and g- cyclodextrins with relative abundances of 95,3 and 2%, respectively.

KEY WORDS:  cyclodextrins, cyclodextrin glycosyltransferase, Bacillus circulans

Chemical characterization of essential oil from seeds of wild and cultivated carrots from Serbia
Acimovic, M., Stankovic, J., Cvetkovic, M., Ignjatov, M., Nikolic, Lj.
Pages 55-60

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ABSTRACT: Seeds from wild carrot (Daucus carota L. ssp. carota) have been used for medicinal purposes since ancient times. Today the oil of its seeds has been proved to possess antinociceptive, antiinflammatory, hypoglycaemic, antidiabetic, antioxidative and anticancer activity. The cultivated carrot (Daucus carota L. ssp. sativus (Hoffm.) Arcang.) is mainly used as a root vegetable, while its seed oil is sometimes employed as a flavouring agent in food products and in the cosmetics industry. In the light of this very different usage, the aim of our investigation was to identify chemical compounds from essential oils of the seeds of these two subspecies of D. carota collected during 2014 in northern Serbia. It is established that wild carrot contains 1.67% of essential oil in the seeds, while cultivated carrot contains 0.55%. In the case of wild-grown carrot, gas chromatographic-mass spectrometric (GC-MS) analyses of seed essential oil show that sabinene (40.9%) and a-pinene (30.1%), followed by ß-bisabolene (6.2%), ß-pinene (5.7%) and trans-caryophyllene (5.3%) are the dominant compounds. The major constituents of essential oil from cultivated carrot seeds are carotol (22.0%), sabinene (19.6%) and a-pinene (13.2%). The mixture of aromadendrene, ß-farnesene and sesquisabinene comprises 8.2%, the content of transcaryophyllene is 5.7% while that of myrcene amounts to 4.7%. Analysis of seeds from both carrots reveals significantly different chemical characteristics of their essential oil, the existence of which influences their different usage.

KEY WORDS:  Daucus carota L. ssp. sativus (Hoffm.) Arcang., Daucus carota L. ssp. carota, GC/MS, sabinene, a-pinene, carotol

Nomenclatural survey of the genus Amaranthus (Amaranthaceae). 4. Detailed questions arising around the name Amaranthus gracilis
Iamonico, D.
Pages 61-68

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ABSTRACT: The present article represents the fourth contribution of a series whose final aim is to gain an understanding of the complicated nomenclature of the genus Amaranthus. The investigation deals with the need to establish the identity of Amaranthus gracilis and related names. On the basis of extensive analysis of the literature, examination of herbarium specimens and field surveys, light has been thrown on a number of complex nomenclatural questions. Amaranthus gracilis is published as a nomen novum for Chenopodium caudatum. According to a recent paper C. caudatum cannot be considered either a member of Chenopodiaceae or an Amaranthus species and is proposed as a nomen rejectendum. Desfontaines’ misinterpretation has never been discussed in depth, and most authors associated A. gracilis with A. viridis. Analysis of the literature has revealed several misapplications. The combinations Euxolus caudatus and Albersia caudata are pro parte synonyms of A. viridis, as are Moquin’s new Euxolus caudatus var. gracilis (lectotype here designated, specimen deposited at P) and the combination Euxolus caudatus var. maximus. The basionym Amaranthus maximus (lectotype here designated, specimen at BM) is a heterotypic synonym of A. caudatus. On the basis of the results obtained, it can be concluded that Amaranthus gracilis has the same type as Ch. caudatum and, if the proposed rejection of this latter name is accepted, then Desfontaines’ name should be rejected too, according to Art. 56.1 of ICN. The name A. major (here lectotypified on a specimen preserved at MPU) is proposed as a heterotypic synonym of A. viridis, the latter name having nomenclatural priority.

KEY WORDS:  Amaranthus viridis, Chenopodium acuminatum, Ch. caudatum, misapplication of names, N.J. Jacquin, typification

Distribution of the genus Galanthus L. (Amaryllidaceae) in Serbia
Jovanovic, F., Obratov-Petkovic, D., Niketic, M., Vukojicic, S.
Pages 69-81

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ABSTRACT:  The distribution of species from the genus Galanthus in Serbia is presented, and notes on their taxonomy and ecology are given in the paper. To date, only two species from the genus are known to be present, namely G. nivalis and G. elwesii. Further research on delimitation of the taxa occurring in Serbia is needed.

KEY WORDS:  Galanthus, Serbia, distribution, ecology, taxonomy

Evidence from multivariate morphometric study of the Quercus pubescens complex in southeast Italy
Di Pietro, R., Di Marzio, P., Medagli, P., Misano, G., Silletti, N.G., Wagensommer, P.R., Fortini, P.
Pages 83-100

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ABSTRACT:  The name Quercus pubescens s.l. encompasses a complex of deciduous oak taxa with mainly southeast- European distribution and a large ecological niche. As the easternmost region of Italy, Apulia is rather isolated from a geographical and physiographical viewpoint and counts the highest number of oak species (10). In the taxonomic and phytosociological literature, the occurrence of several species belonging to the Quercus pubescens collective group is reported for this region. In order to verify if different sets of morphological characters are associated with different taxa, 24 populations of Quercus pubescens s.l. located in different ecological-geographical areas of Apulia were sampled. A total of 367 trees, 4254 leaves and 1120 fruits were collected and morphologically analysed. Overall, 25 morphological characters of oak leaves and fruits were statistically treated using both univariate and multivariate analysis. Nested ANOVA showed that leaves collected from a single tree exhibited a degree of morphological variability higher than that observed when comparing leaves coming from different trees of the same population and from different trees of different populations as well. Almost all the morphological characters analysed exhibited a continuous trend of variation so that none of them can be used as a character to discriminate between populations. Only leaf and fruit “size” and fruit petiole length emerged as slightly discriminating characters. Our results suggest that it is unlikely that more than one species belonging to the Quercus pubescens complex occurs in the Apulia region. Comparison between the Apulian populations and a genetically pure Q. pubescens population coming from a different area (the Molise region) strengthened the assumption as to the existence of a single species that can provisionally be classified under the name of Q. pubescens s.l.

KEY WORDS:  fruit, genetic assignment, leaf, morphometric data, Quercus, statistical analysis, taxonomy

Sphagnum fimbriatum, a species new for the flora of Serbia
Veljic, M., Bukvicki, D., Marin, D.P.
Pages 101-103

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ABSTRACT:  Twenty-four species from the genus Sphagnum have been known up to now in the flora of Serbia. Based on a detailed analysis of herbarium material collected on Mt. Ostrozub, we identified S. fimbriatum Wilson, a species new to the bryoflora of Serbia

KEY WORDS:  Sphagnum fimbriatum, Mt. Ostrozub, Serbia

Corrigenda to “Is it possible to identify Colchicum neapolitanum s.l. and C. autumnale s.l. in vegetative stage? Biometry and flow cytometry approaches” [Botanica Serbica 38 (1): 43-56. 2014]
Fridlender, A.
Pages 105

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ABSTRACT:  Colchicum neapolitanum (Ten.) Ten. subsp. gracile (K. Perss.) Fridl., Botanica Serbica 38(1): 45 (2014), was published as comb. stat. nov. based on C. gracile K. Perss., Bot. Jahrb. Syst. 127(3): 284 (2008). Unfortunately, the name published in 2008 used as a basionym was invalidly published (Art. 8.2, 37.2 ICBN, 2006). Therefore, this new combination was also invalid, as the exceptions listed under Art. 41.8 (ICBN 2012) do not apply. Colchicum gracile K. Perss. was validly published in 2009 and validation of the new combination based on this name is done hereunder.