Renata Iličić

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.

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.

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.

Effect of Indigenous Pseudomonas sp. and Bacillus sp. Strains on Yield and Main Chemical Growth Parameters of Radicchio

Summary Pseudomonas sp. and Bacillus sp. belong to plant growth promoting rhizobacteria which are able to colonize the plants roots and stimulate growth. In this study, the effect of two indigenous plant growth promoting rhizobacterial strains Pseudomonas sp. Q4 and Bacillus sp. Q10 and their mixture (mix Q4+Q10) on content of the main chemical growth parameters (nitrogen, phosphorus, potassium, calcium and magnesium) and the yield of dry biomass of radicchio (Cichorium spp. var. rossa di treviso) aerial parts and root, was investigated. The study was carried out with stagnosol type of soil in pot experiments under semi-controlled conditions in the Institute of Soil Science (Belgrade), in the period from July to October in 2013. Phosphorus was determined by spectrophotometer, potassium - by flame emission photometry and total nitrogen and carbon - using elemental CNS analyzer, while calcium and magnesium were determined by AAS. The data on yield of both aerial parts and root dry biomass of radicchio showed that its treatment with Q4 and Q10 strains, as well as with their mixture, caused noticeably increase in this parameter in relation to the control, whereby the strain Q4 was more effective for aerial parts, while mix Q4+Q10 - for roots. The obtained data on the studied chemical parameters of radicchio root and aerial parts were in total accordance with their yield. Concluding, studied strains have a potential in promoting the biomass yield and main chemical growth parameters of both aerial parts and root of radicchio.

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.

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.