MICROBIOLOGIA BALKANICA th BALKAN CONGRESS OF MICROBIOLOGY & 8th CONGRESS OF SERBIAN MICROBIOLOGISTS, oktobar 2011

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2 CIP Katalogizacija u publikaciji Narodna biblioteka Srbije, Beograd Udruženje mikrobiologa Srbije, Beograd. Knjiga radova (Elektronski izvor) Proceedings / MICROBIOLOGIA BALKANICA th BALKAN CONGRESS OF MICROBIOLOGY & 8th CONGRESS OF SERBIAN MICROBIOLOGISTS, oktobar 2011; (organizator) Udruženje mikrobiologa Srbije, Udruženje medicinskih mikrobiolga Srbije; (urednici: Dragojlo Obradović, Lazar Ranin, Špiro Radulović) Beograd 1 elektronski optički disk (CD-ROM); 12cm Sistemski zahtevi: Nisu navedeni. Nasl. sa naslovnog ekrana. Radovi na engleskom jeziku.- Tekst latinica. Tiraž 600. Abstracts. Registar ISBN Udruženje mikrobiologa Srbije, Beograd. KNJIGA RADOVA / PROCEEDINGS MICROBIOLOGIA BALKANICA th BALKAN CONGRESS OF MICROBIOLOGY & 8th CONGRESS OF SERBIAN MICROBIOLOGISTS, oktobar 2011 Izdaje / Published by: Udruženje mikrobiologa Srbije Nemanjina 6, Beograd, Srbija, tel/fax: , dobradovic@agrifaculty.bg.ac.rs Za izdavača / For Publisher: Dragojlo Obradović, predsednik Udruženja Urednici/Editors: Dragojlo Obradović Lazar Ranin Špiro Radulović ISBN Kompjuterska obrada teksta / Computer Layout: Jelena Zovko Belić Tiraž / Circulation 600 primeraka / 600 copy Umnožavanje / Copying Megaphone d.o.o., Vladimira Rolovića 105, Beograd, Srbija

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4 Identification of Agrobacterium vitis as Causal Agent of Grapevine Crown Gall in Serbia Nemanja Kuzmanović, Katarina Gašić, Milan Ivanović, Anđelka Ćalić, Aleksa Obradović University of Belgrade, Faculty of Agriculture Introduction Grapevine crown gall is the most important and widespread bacterial disease of grapevine (Vitis vinifera L.) throughout the world. The predominant causal agent of this disease is Agrobacterium vitis. Occasionally, tumorigenic strains of A. tumefaciens and A. rhizogenes may also occur on grapevine. In Serbia, symptoms of grapevine crown gall were observed for the first time in 1962 in the area of Trstenik on the cultivar Cardinal imported from Italy. However, this disease and causal agent were not studied extensively in the last 30 years in our country. Aim During 2010 and 2011 an outbreak of crown gall disease was observed on 3-5 years old grapevine on various cultivars throughout Serbia. Large aerial tumors were visible on canes, above or around the grafting point, in most cases completely girdling the trunk. The aim of this study was to isolate and identify causal agent of the disease using standard biochemical and physiological test as well as molecular based techniques. Material and methods Isolation was performed on Yeast Mannitol Agar (YMA) medium. For species/biovar determination, the following tests were performed: fluorescence on King s B medium, Gram and oxidase reaction, growth in 2% NaCl and at 35 C, 3-ketolactose production, acid clearing on PDA plus CaCO 3, ferric ammonium citrate test, motility at ph 7.0, pectolytic activity at ph 4.5, citrate utilization, production of acid from sucrose and alkali from tartarate. The strains were also differentiated to the species/biovar level using multiplex PCR assay targeting 23s rrna gene sequences. Pathogenicity genes vird2 and virc, located on plasmid, were detected using PCR assays with specific primers. Pathogenicity was confirmed by artificial inoculation of three plants per strain of grapevine cv. Cabernet Franc and local tomato cultivar (Lycopersicon esculentum L.). Results and Conclusion From the transformed tissue, white, circular and glistening bacterial colonies were isolated on YMA medium. Eight non-fluorescent, Gram-negative and oxidase positive strains were selected for further identification. The strains grew at 35ºC and in nutrient broth with 2% NaCl. They were negative in 3-ketolactose, acid clearing PDA- CaCO 3, and ferric ammonium citrate tests; non motile at ph 7.0; pectolytic at ph 4.5; utilized citrate; produced acid from sucrose and alkali from tartarate. Based on the results of physiological and biochemical tests the strains were assigned as Agrobacterium vitis which was also confirmed by PCR analysis of 23s rrna gene. PCR assays with vird2 and virc gene-specific primers yielded the products of expected size, confirming that the strains harbored the plasmid responsible for pathogenicity. Typical tumors developed at the inoculation sites of tomato plants 3 weeks after inoculation and on grapevine plants 3 weeks later. No symptoms were observed on control plants. Bacteria were reisolated from tumorigenic tissue and determined as pathogenic A. vitis by PCR. This is the first report of A. vitis identified as the causal agent of grapevine crown gall in Serbia. Keywords: grapevine crown gall, Agrobacterium vitis, plasmid, pathogenicity This study was supported by the Serbian Ministry of Education and Science, through the Project III46008.

5 Identification of Agrobacterium vitis as Causal Agent of Grapevine Crown Gall in Serbia Kuzmanović Nemanja, Gašić Katarina, Ivanović Milan, Ćalić Anđelka, Obradović Aleksa University of Belgrade, Faculty of Agriculture, Department of Plant Pathology, Belgrade, Serbia Introduction Grapevine crown gall is the most important and widespread bacterial disease of grapevine (Vitis vinifera L.) throughout the world. The predominant causal agent of this disease is Agrobacterium vitis. Occasionally, tumorigenic strains of A. tumefaciens and A. rhizogenes may also occur on grapevine. In Serbia, symptoms of grapevine crown gall were observed for the first time in 1962 in the area of Trstenik on the cultivar Cardinal imported from Italy. However, this disease and causal agent were not studied extensively in the last 30 years in our country. During 2010 and 2011 a serious outbreak of crown gall disease was observed on 3-5 years old grapevine on various cultivars throughout Serbia. Large aerial tumors were visible on canes, above or around the grafting point, in most cases completely girdling the trunk (Figure 1). The aim of this study was to isolate and identify causal agent of the disease using standard biochemical and physiological test as well as molecular based techniques. A B Figure 1. Grapevine crown gall. Trunk of grapevine girdled by tumor (A). Longitudinal section of tumor (B). Material and methods Results Isolation of bacterial strains Samples were collected from commercial vineyards located in the South Banat District in Serbia. Fragments of fresh tumor tissue were incubated in sterile distilled water for 2 hours to allow the bacteria to diffuse into the liquid. Loopfuls of tissue suspensions were streaked on yeast mannitol agar (YMA) medium. Plates were incubated 3-5 days at 28ºC. Representative colony types were purified and maintained on potato dextrose agar (PDA) supplemented with 0.4% CaCO3. White, circular and glistening bacterial colonies were developed on YMA medium after 3 days of incubation. Pigmentation and morphology of selected bacterial colonies were similar on PDA supplemented with 0.4% CaCO3 (Figure 2). Five strains originating from five different plants /tumors were selected for identification. PCR assays with vird2 and virc gene-specific primers yielded 224 and 414-bp fragments, respectively, confirming that strains harbored the plasmid responsible for pathogenicity (Figure 3). Tumorigenic (Ti) plasmid detection Selected strains were analyzed by PCR with A/C and VCF3/VCR3 primers corresponding to the vird2 and virc pathogenicity genes located on tumorigenic plasmid. Differentiation to biovar/species level For species/biovar determination, the following tests were performed: fluorescence on King s B medium, Gram and oxidase reaction, growth in 2% NaCl and at 35 C, 3-ketolactose production, acid clearing on PDA plus CaCO3, ferric ammonium citrate test, motility at ph 7.0, pectolytic activity at ph 4.5, citrate utilization, production of acid from sucrose and alkali from tartarate. The strains were also differentiated to the species/biovar level using multiplex PCR assay targeting 23s rrna gene sequences. Using this method, identification of four Agrobacterium species/biovars is enabled. Pathogenicity assay Pathogenicity assay was performed by inoculation of grapevine cv. Cabernet Franc and local tomato cultivar (Lycopersicon esculentum L.). The plants were inoculated on the stem by pricking 1-3 times through a drop of inoculum (108 CFU/ml). Sterile distilled water was used as a negative control. Inoculated plants were maintained in a greenhouse at 24 ± 3 C. Figure 3. PCR analysis of the strains. VirC primer pair amplified 414-bp product of all strains. Lane M, marker (MassRuler Low Range DNA Ladder, Fermentas, Lithuania); Lane 1 - KFB 096 (referent strain of A. tumefaciens); Lane 2 - KFB 098 (referent strain of A. rhizogenes) Lane 3 - KFB 099 (referent strain of A. vitis); Lane bacterial strains used in this study; lane W negative control. Figure 2. Pigmentation and colony morphology of selected strains on PDA supplemented with 0.4% CaCO3. All strains were non-fluorescent, oxidase positive, grew at 35ºC and in nutrient broth with 2% NaCl. They were negative in 3-ketolactose, acid clearing PDACaCO3, and ferric ammonium citrate tests; non motile at ph 7.0; pectolytic at ph 4.5; utilized citrate; produced acid from sucrose and alkali from tartarate. Based on the results of physiological and biochemical tests the strains were assigned as A.vitis. This was also confirmed by PCR analysis of 23s rrna gene. Typical tumors developed at the inoculation sites of tomato plants 3 weeks after inoculation and on grapevine plants after 6 weeks (Figure 4). No symptoms were observed on control plants. A B C Figure 4. Pathogenicity assay. Grapevine (A) and tomato (B) plants inoculated with strain used in this study. Tomato plant inoculated with sterile distilled water (C). Conclusion Crown gall disease was sporadically observed in vineyards in Serbia in previous years, and did not cause significant damage. Therefore, the etiology of this disease was not studied in detail. This is the first report of A. vitis determined as the causal agent of grapevine crown gall in Serbia. Bacterial strains of A. vitis were identified and differentiated to the species level combining classical bacteriological and molecular methods. This research was supported by the Serbian Ministry of Education and Science, through the Project III46008.