Jelica Balaž

First Report of Pseudomonas syringae pv. syringae Causing Bacterial Leaf Spots of Oil Pumpkin (Cucurbita pepo) in Serbia

Oil pumpkin (Cucurbita pepo L.) is commonly used for oil production, mainly in central and eastern Europe (1). In Serbia, it grows only in the north (Vojvodina Province), up to 1,500 ha. In June 2008, typical bacterial spot symptoms (dark green, water-soaked, transparent and greasy spots with yellow margins) were observed for the first time, cultivated at the experimental fields near Backi Petrovac. Since then, bacterial spots were regularly observed on oil pumpkin in the beginning of the growing seasons and during rainy weather, with disease incidence ranging from 5 to 20%. Bacteria isolated from 40 diseased leaves formed white, round, convex, and mucoid colonies on nutrient sucrose agar (NSA). Eight representative strains were aerobic, gram-negative, non-spore-forming rods. All strains produced fluorescent pigment and catalase. In levan-oxidase-potato rot-arginine dihydrolase-tobacco hypersensitivity (LOPAT) tests (3), they induced a hypersensitive reaction in tobacco leaves, did not cause soft rot of potato tubers, and were positive for levan and negative for oxidase and arginine dihydrolase. According to the LOPAT profile, they were classified in the Ia subgroup of pseudomonads (3). Strains hydrolyzed aesculin, but were unable to hydrolyze starch or reduce nitrates to nitrites. Negative reactions were obtained with hydrogen sulfide and indole. Reactions were identical to those of reference strain Pseudomonas syringae pv. syringae CFBP 1582, which was included in all biochemical, physiological, and molecular tests for comparison. To identify the pathogen, PCR and DNA sequencing were employed. Fragments of 752 bp for the syrB gene and 1,040 bp for the syrD gene were amplified from all strains, using B1/B2 and SyD1/SyD2 primer sets, respectively (2). The pathogenicity was tested on seeds and seedlings of oil pumpkin cv. Olinka. Strains were grown for 48 h on nutrient broth (NB) at 28°C and bacterial suspensions of ~108 CFU ml-1 were used for inoculations. Sterile water was used as negative control. Seeds (at the BBCH-1-0 stage) allowed to imbibe water were wounded by needle, immersed in the bacterial suspensions, and maintained in humid petri dishes to allow symptom development. The cotyledons of seedlings at the BBCH-10 stage were inoculated by hypodermic needle and potted plants were maintained at 25 ± 1°C and 75% relative humidity. Symptoms, including dark green, water-soaked spots, appeared 5 to 7 days after inoculation of both seeds and seedlings. The bacterium was re-isolated from spots of all seeds and seedlings tested, fulfilling Kochs postulates (the identity of re-isolated strains was confirmed by pathogenicity, morphology, and biochemical features). No symptoms were observed on controls. 16S rDNA amplicons obtained from representative strain Tk21 and re-isolated strain Tk21R with fD1/rD1 primers (4) were sequenced and deposited in GenBank under accession nos. KF305578 and KF735064, respectively. The sequences showed 100% similarity to each other and P. syringae pv. syringae from pepper (KC816630.1) (China), Ficus carica (JQ071937) (Serbia), and culture-collection ICMP:3023 (HM190217). On the basis of the symptoms, biochemical tests, and 16S rDNA sequence homology, the pathogen was identified as P. syringae pv. syringae. To our knowledge, this is the first report of P. syringae pv. syringae causing bacterial leaf spot on oil pumpkin in Serbia. References: (1) J. Berenji et al. Oil pumpkin Cucurbita pepo. Monography. IFVC, Novi Sad, 2011. (2) K. Gasic et al. Pestic. Phytomed. 27:219, 2012. (3) R. A. Lelliott et al. J. Appl. Bact. 29:470, 1966. (4) W. G. Weisburg et al. J. Bacteriol. 173:697, 1991.

Elaboration of methods for detection of Pseudomonas savastanoi pv. phaseolicola on bean seeds

Pseudomonas savastanoi pv. phaseolicola detection on artificially inoculated bean seeds was investigated. The method of the International Seed Federation - ISF (2006) was used. It includes bacteria extraction from seeds, isolation on semiselective media and checking the pathogenicity of investigated isolates. For verification of results, quick new methods of investigation were used (ELISA test and PCR). The results show that semiselective media MT (Milk Tween Agar) and MSP (Modified Sucrose Peptone Agar) can be appropriate for isolation of this bacterium. Pathogenicity of the investigated isolates was confirmed on cotyledon leaves of bean. ELISA test and PCR confirmed that all investigated isolates and reisolates belong to the bacterium P.s.pv. phaseolicola.

The application of levulinic acid and 5-nitro-2-furylmethylene diacetate in the total synthesis of some novel biologically active (5-nitro-2-furyl)azomethines

SummaryThe syntheses andin vitro antibacterial and antifungal evaluation of certain (5-nitro-2-furyl)azomethines with different heterocyclic nuclei are described.ZusammenfassungEs wird die Synthese und diein-vitro-antibakterielle und antifungale Wirksamkeit für bestimmte (5-Nitro-2-furyl)azomethine mit verschiedenen heterocyclischen Kernen beschrieben.

ETIOLOGY OF BACTERIAL CANKER ON YOUNG SWEET CHERRY TREES IN SERBIA

Bacterial canker of sweet cherries caused by Pseudomonas syringae was studied in the Vojvodina Province (region of Serbia) with the aim of better understanding disease etiology and epidemiology. During the last decade, bacterial canker has become a recurrent problem, especially in young plantations characterized by intensive production and lack of effective control measures. Among the 24 isolates collected from two localities (Selenca and Gornji Tavankut), classical bacteriology tests, pathogenicity, as well as genetic methods based on PCR for syrB gene and DNA sequencing with four housekeeping genes (MLSA), were performed allowing identification of 10 isolates as P. syringae pv. syringae and 14 isolates as P. syringae pv. morsprunorum race 1. In both pathovars, genetic groups were homogenous, confirming earlier research findings and contributing to the extant knowledge of its geographic distribution.

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.

Application of semi-selective mediums in routine diagnostic testing of Pseudomonas savastanoi pv. phaseolicola on common bean seeds

Halo blight, caused by Pseudomonas savastanoi pv. phaseolicola (Psp), is considered to be an important bacterial disease on common bean (Phaseolus vulgaris L.) in Serbia. Use of pathogen-free seeds is one of the most effective control measures against this disease. The aim of this study was to evaluate a detection method for Psp on untreated common bean seeds (23 genotypes) from commercial crops grown within Serbia. Detection of this pathogen was made by plating onto the modified sucrose peptone (MSP) and Milk Tween (MT) semi-selective mediums from soaked whole common bean seed. Colonies growing on the MSP medium were light yellow, convex and shiny, whereas on the MT medium, they were creamy white, flat and circular. The pathogenicity of the obtained strains was confirmed by the inoculation of germinated bean seed. The isolates recovered from the seed assay were further confirmed to be Psp by using both Enzyme-linked immunosorbent assay (ELISA) and polymerase chain reaction (Nested-PCR) detection methodologies. The International Seed Testing Association (ISTA) method selected for this work was found to be effective in detecting the presence of Psp in common bean seed. The bacterium Psp was detected in only two of the 23 seed samples analyzed by this method, which shows that the bacterium is not widespread in Serbia.

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.