Tatjana Popovic

Polyphenolic Characterisation of Vranac, Kratosija and Cabernet Sauvignon (Vitis vinifera L. cv.) Grapes and Wines from Different Vineyard Locations in Montenegro

In Montenegro, red wines are produced predominantly, and Vranac accounts for nearly 80% of these wines, followed by Kratosija and Cabernet Sauvignon. In order to characterise polyphenols in red varieties, grapes were sampled from representative vineyards at harvest time during 2011 and 2012. The content and distribution of extractable anthocyanins, low-molecular mass proanthocyanidins (LMP) and highmolecular mass proanthocyanidins (HMP) in the seeds and skins of the grape berries were evaluated by applying a five-day extraction method using ethanol:water (12:88) as extraction solvent. On average, the highest content of LMP (2 006 and 1 690 mg/kg of grape fresh mass in years 2011 and 2012 respectively), HMP (2 705 and 2 805 mg/kg in years 2011 and 2012 respectively) and anthocyanins (1 035 mg/kg in the year 2011) was found in the Cabernet Sauvignon grapes. The highest content of anthocyanins (1 113 mg/kg in the year 2012) and the lowest content of LMP (1 103 and 846 mg/kg in years 2011 and 2012 respectively) was found in Vranac grapes. Kratosija grapes had the lowest anthocyanin content (456 and 517 mg/kg in years 2011 and 2012 respectively), and levels of LMP were similar to Vranac. The percentage distributions of LMP between skins and seeds were 34:66, 39:61 and 49:51, whereas the distributions of HMP between skins and seeds were 67:33, 62:38 and 64:36 for Vranac, Kratosija and Cabernet Sauvignon respectively. All varieties had more LMP in the seeds and more HMP in the skins of the grapes. The results obtained are important to better understand the polyphenolic potential of Montenegrin red grape varieties.

Molecular assessment of genetic diversity of Xanthomonas arboricola pv. juglandis strains from Serbia by various DNA fingerprinting techniques

The purpose of the present study was to investigate the genetic diversity of X. arboricola pv. juglandis strains in Serbia. This bacterium is the causal agent of walnut blight and is also associated with apical necrosis of immature walnut fruits. Although walnut blight is long known and widespread in Serbia, a systematic strain diversity study of Xanthomonas arboricola pv. juglandis strains from different regions in Serbia has not been performed. The objectives of this work were to examine the molecular diversity and its possible biological significance of 59 isolates of X. arboricola pv. juglandis collected from different geographic locations in Serbia. Genomic variability was assessed by using repetitive PCR, SpeI macrorestriction analysis of genomic DNAs by pulsed-field gel electrophoresis (PFGE) and partial sequencing of the gyrB gene. Molecular analyses showed substantial genetic diversity among strains and existence of diverse populations of X. arboricola pv. juglandis in Serbia.

Cultivated and wild plantain (Plantago major) as a host of Stolbur phytoplasma in Serbia

The wild plantain (Plantago major) is an important medicinal plant. Symptoms suggestive of phytoplasma diseases were observed in infected plantain plants in Serbia. A new disease on P. major has symptoms of reduced leaf size, leaf reddening and crinkling, and occasionally rolling of flowers and early drying up. This disease was found first on the plantain plantation in Pancevo locality, but later has been found in some other localities in Serbia (Kovin, Vrdnik). Restriction fragment length polymorphism (RFLP) analysis of amplification products of 1.2 kb, obtained in nested PCR with R16F2n/R16R2 primer pair after amplification with P1/16S-Sr primers, in 24 from 26 symptomatic plants indicated the presence of phytoplasma from the 16SrXII-A subgroup. Plantain plants collected from all three affected localities in Serbia were determined to be hosts of this phytoplasma. This is the first report of the natural occurrence of Stolbur phytoplasma in cultivated and wild P. major in Serbia.

First report of Xanthomonas campestris pv. campestris as the causal agent of black rot on oilseed rape (Brassica napus) in Serbia.

In September 2010, leaves of oilseed rape (Brassica napus L.) with v-shaped, necrotic lesions on the leaf margins surrounded by yellow halos were collected. Symptoms were observed on the domestic cultivar Slavica (IFVC, Novi Sad) located in the Bačka region, Vojvodina, Serbia, from a 3-ha field. Average disease incidence on 3-month-old plants was 45% (15 to 75%). Diseased leaves were rinsed in sterilized distilled water (SDW) and dried at room temperature for isolations. Leaf sections taken from the margin of necrotic leaf tissue were macerated in SDW and the extract was streaked onto yeast extract-dextrose-calcium carbonate (YDC) agar. Plates were incubated at 28°C for 3 days. Colonies were yellow, translucent, circular, and raised. Ten representative strains tested further were all gram-negative, catalase-positive, and oxidase-negative. The partial 16S rDNA sequence of a representative strain, TUr1, was amplified using primers fD1 and rD1 (2), and determined using the IMGGI SeqService facility in Belgrade. The 1,510-bp 16S rDNA sequence of TUr1 was compared to that of known strains in the NCBI GenBank database, and showed greatest similarity with that of Xanthomonas campestris pv. campestris (Xcc) strains ATCC 33913 and B100 (99% homology). Pathogenicity of 10 strains grown for 48 h on YDC at 28°C was completed using each of three methods: spraying a bacterial suspension (108 cfu/ml) onto the leaf surfaces of oilseed rape plants, stabbing the major veins of each of the first two true leaves with the tip of a sterile toothpick that had been dipped into a colony of the appropriate strain, and immersing cotyledons of the plants into a bacterial suspension (108 cfu/ml). All three tests were performed on 4-week-old oilseed rape plants of the cultivar Slavica. SDW was used for the negative control treatment for each method of inoculation. Reference strain Xcc NCPPB 1144 was used as a positive control treatment. Tests plants (two for each method of inoculation and each bacterial strain or control treatment) were maintained in a greenhouse at 25 ± 1°C and 80% relative humidity by keeping the plants in plastic bags. Two control plants for each of the negative and positive control treatments for each inoculation method were also enclosed in separate plastic bags. The bacterial strains and reference strain caused yellow lesions on inoculated plants that turned necrotic starting about 7 days after inoculation (DAI). The spots coalesced within 21 DAI to form necrotic areas. Plants inoculated with SDW remained symptomless. Reisolations were done onto YDC as described above. Reisolated strains showed the same colony morphology as described above. The bacterial strains grew at 35°C; produced levan from sucrose, hydrogen sulfide, and indole; did not reduce nitrate; hydrolyzed Tween 80; starch, gelatin, and aesculin; did not show tolerance to 0.10 and 0.02% triphenyl-tetrazolium chloride; and produced acid from d-arabinose, arginine, dulcitol, galactose, d-glucose, maltose, mannose, sorbitol, sucrose, and xylose (1). All strains tested by Plate Trapped Antigen-ELISAs (ADGEN Phytodiagnostics, Neogen Europe Ltd., Scotland) reacted with Xcc-specific polyclonal antibodies. Based on these tests, the strains were identified as Xcc. To our knowledge, this is the first report of this pathogen causing black rot of oilseed rape in Serbia. References: (1) T. B. Adhikariand and R. Basnyat. Eur. J. Plant Pathol. 105:303, 1999. (2) W. G. Weisburg et al. J. Bacteriol. 173:697, 1991.

A blight disease on highbush blueberry associated with Macrophomina phaseolina in Serbia

Abstract Unusual blight-like symptoms appeared on highbush blueberry plants in Serbia during August 2015 and infected plants showed browning and reddening of leaves, drying of foliage and brown discolouration of internal vascular stem tissues. The objective of this study was to isolate and confirm a causal agent of the disease. Five diseased blueberry plants (2-year-old), with visible brown discolouration in the wood, were collected for isolation on potato dextrose agar (PDA). Morphological analysis of the selected fungal isolates showed the presence of abundant black, round to oblong, or irregularly shaped microsclerotia immersed in the PDA. Dark, globose pycnidia formed on water agar with an initially hyaline, granular content and single-celled conidia, indicating the presence of plant pathogenic fungus Macrophomina phaseolina associated with symptomatic plant tissues. Pathogenicity was confirmed on potted blueberry plants based on the initial symptoms of leaves turning yellowish to brown at the leaf edges, followed by the defoliation of leaves of the inoculated stems. Discolouration of vascular tissues was also observed on transverse sections of inoculated stems. The pathogen M. phaseolina was confirmed using molecular analysis of the ITS1-5.8S-ITS2 region of rDNA and a part of the TEF-1α gene region. This is the first report of M. phaseolina causing a blight disease on highbush blueberry in Serbia. The study should help in elucidating disease symptomatology and provide information on the risk which this fungus could pose in blueberry production.

Characterization of Xanthomonas hortorum pv. pelargonii Isolated from Geranium in Serbia

Geranium leaves and stems with symptoms of bacterial blight were collected from commercial greenhouses during the last decade in Serbia. In total, 17 isolates with colony morphology typical for the genus Xanthomonas were characterized with pathogenicity, biochemical, serological, and molecular assays. All 17 isolates reacted positive in a polymerase chain reaction (PCR) using XcpM1 and XcpM2 primers specific for Xanthomonas hortorum pv. pelargonii. In pathogenicity tests on Pelargonium zonale (leaf and stem inoculation), all isolates caused typical symptoms on leaves starting 2 days after inoculation as sunken, water-soaked, irregular lesions, and 6 to 8 days after inoculation on stems as necrotic lesions also showing yellow exudate. Symptoms resulted in general wilting of inoculated plants 20 days after inoculation. Selected phenotypic tests indicated that all isolates showed the same results as described for the bacterium X. hortorum pv. pelargonii. Repetitive sequence-based PCR typing using BOX and ERIC revealed that all isolates showed two fingerprinting profiles but (GTG)5 and REP did not reveal differences. Multilocus sequence typing of partial sequences of rpoD, dnaK, fyuA, and gyrB genes of tested isolates and sequences obtained from GenBank of Xanthomonas pathovar pathotype strains did not reveal genetic variability among the isolates, showing the same gene sequence pattern.

First Report of Colletotrichum clavatum Causing Quince Anthracnose in Serbia

Quince (Cydonia oblonga Mill.) tree is traditionally grown in Serbia. The fruits are used for compote, marmalade, and brandy production. In December 2012, quince fruits cv. Leskovacka with symptoms of postharvest anthracnose were collected in a storage facility in the area of Sabac, western Serbia. The symptoms were observed on fruits approximately 2 months after harvest. The incidence of the disease was about 3%, but the symptoms were severe. Affected fruits showed sunken, dark brown to black lesions with orange conidial masses produced in black acervuli. Small pieces (3 to 5 mm) of necrotic tissue were surface sterilized for 1 min in 1% NaOCl, washed twice with sterile distilled water, and placed on potato dextrose agar (PDA). Macroscopic and microscopic morphology characteristics of three isolates were observed after growth on PDA for 7 days at 25°C under a 12-h photoperiod. Fungal colonies developed white to gray dense aerial mycelium with orange conidial masses in the center of the colony. Conidia were hyaline, aseptate, clavate with rounded distal apices, 15.2 (12.8 to 16.8) × 4.5 (4.0 to 5.2) μm (mean L/W ratio = 3.3, n = 100). Morphological characteristics are consistent with the description of Colletotrichum clavatum (2). Fungal isolates were also characterized by sequencing of the internal transcribed spacer (ITS) rDNA region using ITS1/IT4 primers and β-tubuline 2 gene using T1/T2 primers. The nucleotide sequences were deposited in GenBank (ITS Accession Nos. KF908866, KF908867, and KF908868; β-tubuline 2 gene KF908869, KF908870, and KF908871). BLAST analyses of ITS and β-tubuline 2 gene sequences showed that isolates from quince were 100% identical to other C. clavatum in GenBank (ITS JN121126, JN121130, JN121132, and JN121180; β-tubuline 2 gene JN121213 to 17, JN121219, JN121228, JN121261 to 62, and JN121266 to 69), thus confirming the morphological identification. To fulfill Kochs postulates, asymptomatic fruits of quince cv. Leskovacka (five fruits per isolate) were surface sterilized with 70% ethanol, wounded with a sterile needle, and inoculated with 50 μl of a spore suspension (1 × 106 conidia/ml). Five control fruits were inoculated with 50 μl of sterile distilled water. The experiment was repeated twice. After 10 days of incubation in plastic containers, under high humidity (>90% RH) at 25°C, typical anthracnose symptoms developed on inoculated fruits, while control fruits remained symptomless. The isolates recovered from symptomatic fruits showed the same morphological features as original isolates. C. clavatum previously indicated as group B (3), or genetic group A4 within the C. acutatum sensu lato complex (4), is responsible for olive anthracnose in some Mediterranean countries (1,2), and has been reported as causal agent of anthracnose on a wide range of other hosts including woody and herbaceous plants, ornamentals, and fruit trees worldwide (4). To our knowledge, this is the first report of C. clavatum in Serbia, and the first report of quince anthracnose caused by this pathogen in Europe. Anthracnose caused by C. clavatum can endanger the production and storage of quince in the future, and may require investigation of new disease management practices to control this fungus. References: (1) S. O. Cacciola et al. J. Plant Pathol. 94:29, 2012. (2) R. Faedda et al. Phytopathol. Mediterr. 50:283, 2011. (3) R. Lardner et al. Mycol. Res. 103:275, 1999. (4) S. Sreenivasaprasad and P. Talhinhas. Mol. Plant Pathol. 6:361, 2005.

The effectiveness of potassium phosphite and captan mixture in controlling Venturia inaequalis in apple orchards

Venturia inaequalis is a common apple disease in Serbia and its intensity depends on weather conditions. The most successful method of combating apple scab is to use a lower dose of an active ingredient and increase its effectiveness by adding substances that do not cause environmental damage. The effectiveness of a fungicide mixture of captan and potassium phosphite against V. inaequalis was investigated in apple orchards in 2008 and 2009. A relevant EPPO standard method was used in all trials. Captan 50 WP was used as a standard fungicide. In order to determine the exact conditions marking the onset of apple infection, key meteorological conditions were monitored in the orchards using an automated iMetos weather station and a Lufft electronic instrument. The results showed high efficacy of the tested fungicides which remained similar in both years of investigation. The intensity of leaf infection after treatment with the mixture of captan and potassium phosphite was 0.7-2.4%, while fruit infection was 0.5-3.0%. The efficacy of this mixture ranged from 96.3-97.9% on leaves and 87.4-98.7% on fruits. The standard fungicide Captan 50 WP showed an efficacy ranging from 95.3-96.7% on leaves and from 87.8-99.3% on fruits. No statistically significant differences were found between the fungicide tested and the standard fungicide. [Projekat Ministarstva nauke Republike Srbije, br. BT31018 i br. III46007]

Efficacy of Chemical Weed Control in Potato (Solanum tuberosum L.)

In this paper the results of efficiency of combined application of six herbicides in weed control in potato were presented. The study was done during 2007 and 2008 in Kolasin, on acid brown soil, at an altitude of about 900 m. In the experiment the following herbicides were examined: S-metalachlor, Bentazone, Acetohlor, Flurochloridone, Metribuzin and Dimetenamid-P. The study was conducted in the Kennebec variety crop. All applied herbicides had satisfactory effect in decreasing number and biomass of weeds. As the most effective variants in two-year average, Sencor 70 WP and Genius were expressed (95 and 94% for the number and 92 and 88.8% for weed biomass) and the weakest effect had combination of Dual Gold 960 EC + Basagran (82.3; 69.4, respectively). In all investigated combinations of herbicides significantly higher tuber yield was achieved comparing to the control. The highest yield of tubers was measured in variant where Acenit 800 EC was applied - 33 t ha -1 , while the lowest yield had the control variant - 18.4 t ha -1 .