Genetic Diversity of Brenneria Nigrifluens Strains in North of Iran
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GENETIC DIVERSITY OF BRENNERIA NIGRIFLUENS STRAINS IN NORTH OF IRAN DOI: 10.2478/v10298-012-0015-8 Cercetări Agronomice în Moldova Vol. XLV , No. 2 (150) / 2012 GENETIC DIVERSITY OF BRENNERIA NIGRIFLUENS STRAINS IN NORTH OF IRAN (MARGIN OF CASPIAN SEA) A. JAMALZADE1*, M. SHAMSBAKHSH1, H. RAHIMIAN2 *E-mail: [email protected] Received March 12, 2012 ABSTRACT. Brenneria nigrifluens, the the strains were different from each other cause of shallow bark canker of Persian and were only useful for preliminary walnut trees (Juglans regia L.), has become grouping of the isolates. The groups of fairly widespread in Iran in recent years. It strains were differentiated by their rep-PCR is regarded as a great threat to walnut fingerprints and on which basis they were production. To determine the diversity of B. placed in six groups in similarity level 95%. nigrifluens strains, sixty strains of the causal Cluster analysis was performed using bacterium were isolated from bark samples NTSYSpd software. The results of these of infected walnut trees collected from studies demonstrated that the populations of Mazandaran, Guilan and Golestan provinces B. nigrifluens in North of Iran are and were studied. The physiological and genetically heterogeneous. The results can biochemical characteristics, electrophoretic be used in selection of disease management patterns of total cell proteins and rep-PCR strategies. generated DNA fingerprints of B. nigrifluens strains were compared. Strains Key words: Shallow bark canker; appeared to be more or less similar in Persian walnut; REP-PCR. phenotypic characteristics. Less than 15% of the strains differed in a few phenotypic INTRODUCTION features such as the ability in production of H2S from peptone, hydrolysis of esculin, One of the important bacterial levan production, arginine dehydrolase, nitrate reduction, indol production and diseases that affect plants of Persian methyl red reaction. These differences did walnut (Juglans regia L.) and can not show any special distribution and provoke significant reduction in therefore was not suitable for classifying the walnut and timber production is strains into distinct groups. The shallow bark canker incited by electrophoretic patterns of cell proteins of Brenneria nigrifluens (Wilson et al., 1 Department of Plant Pathology, Faculty of Agriculture, Tarbiat Modares University, Tehran, Iran 2 Department of Plant Protection, University of Agriculture Sciences and Natural Resources, Sari, Iran 57 A. JAMALZADE, M. SHAMSBAKHSH, H. RAHIMIAN 1957; Hauben et al., 1998). First Up to now different methods were reported in California (Wilson et al., used for identification of B. 1957), The disease was also recorded nigrifluens. For example use of from Spain (Lopez et al., 1994), bacteriofages (Zeitoun and Wilson, several locations in Italy (Saccardi et 1966), study of Serological properties al., 1998; Morone et al., 1998; (Harighi and Rahimian, 1997; Yousefi Scortichini, 1999; Carella et al., 2003; Kopaei et al., 2004), study of Loreti et al., 2005) and France electrophoretic profiles of total cell (Menard et al., 2004). In Iran this proteins (Harighi and Rahimian, 1997; disease has been reported from Scortichini, 1999; Yousefi Kopaei et different regions including al., 2004), Study of fat acids (Wells et Mazandaran (Rahimian, 1989; Harighi al., 1994; Scortichini, 1999) and and Rahimian 1997), Fars and molecular methods for example RFLP Kohgiluyeh and Boyerahmad (Yousefi (Thoth et al., 2001) and Rep-PCR Kopaei et al., 2004), Kerman (Moretti et al., 2007). (Baradaran and Ghasemi, 2004), Determination of genetic Kordestan (Harighi, 2006) and Guilan diversity of a microorganism is and Golestan (Jamalzade et al., important in breeding programs in the 2009). This bacterium identified in search for tolerant or resistant Korea associated with Xanthomonas germplasms (Norelli et al., 1984). axonopodis pv. Pruni that cause peach More recently tree families of & Japanese plum shot hole (Choi et repetitive sequences including al., 2000). In Argentina this pathogen repetitive exteragenic palindromic induces Artichoke violet necrosis (REP) sequence, enterobacterial (Soto, 1997). Also B. nigrifluens in repetitive intergenic consensus Iran isolated from sunflower; but did (ERIC) sequence and BOX element not prove its pathogenisity (Versalovic et al., 1994) which are (Hassanzadeh and Madjidieh- short repetitive DNA sequences have Ghasemi, 1989). been used to design universal PCR The disease is mainly primers that generate highly characterized by shallow, irregular reproducible, strain-specific shaped canker in the bark of the trunk fingerprints that can differentiate and scaffold branches with dark bacterial strains below the level of watery exudates which stained the species (Little et al., 1998). Use of the affected trunk and limb. By removal respective primer (s) in PCR could of phelloderm extensive necrosis of lead to the selective amplification of the underlying tissues were observed. distinct genomic regions located In some cases, necrosis extended to between REP, ERIC or BOX cambium and outer sapwood. It can sequences. provoke sever damage in young With attention to increasing nursery plants (Saccardi et al., 1998) trend of this disease in Iran as well as and on adult trees (Piccirillo, 2003). little knowledge about genetic 58 GENETIC DIVERSITY OF BRENNERIA NIGRIFLUENS STRAINS IN NORTH OF IRAN relationship among isolates of B. Hypersensitive reaction in tobacco nigrifluens, the objective of this study (Klement et al., 1964), Arginine was to investigate of genetic diversity dehydrolase (Thornely, 1960), Methyl-red of B. nigrifluens strains isolated from reaction and starch hydrolysis (Cowan, walnut growing regions in North of 1974), Tween 20 hydrolysis (Misaghi and Grogan, 1970), Levan formation, potato Iran (margin of Caspian sea) by using soft rot, Gelatin and esculin hydrolysis, Rep-PCR. catalase, H2S production from peptone, indol production, reducing substance from MATERIALS AND METHODS sucrose, nitrate reduction, urease production, tolerance to 5% NaCl and Sampling and isolation. More than growth at 36 and 39°C (Schaad et al., 100 samples of walnut branches and trunk 2001). bark with symptoms of shallow bark Protein extraction and SDS- canker were collected from margin of PAGE. For protein extract, bacterial Caspian Sea (Guilan, Golestan and strains grown on NAS for 24 h at 27-28°C Mazandaran provinces). For bacterial were suspended in 1ml sterile distilled isolation, the bark surrounding the canker water to mid log phase (optical density at was removed with a flame sterilized 600 nm [OD600], 1). knife. Small pieces of tissue, collected In order to lyses of cells, 200μl with a scalpel at the edge of the cankers, extraction buffer (125mM Tris-HCl [PH were immersed in a few drops of sterile 6.8], 4% sodium dodesyl sulfate, 2% water contained in plates. After 2-3 h, mercaptoethanol), added to cells affected tissues were ground in the plates suspension and then they were placed in using steel pestles. A loopfull of the boiling water for 5 min. After homogenate was streaked onto modified centrifugation at 1300 rpm for 10 min, 40 Eosin Methilen Blue Agar (EMB-Agar) μl of the supernatant was loaded onto a containing 1% glycerol and 5% Yeast column. A constant current of 25 mA was extract and incubated at 27±1°C. The used to run gel for 3.5 h. The procedure prevalent bacterial colonies and those was performed according to (Laemmli, similar in appearance to B. nigrifluens 1970) using a 5% staking gel (PH 6.8) were selected and purified on NA and a 10% running gel (ph 8.8). Gel were amended with 5% sucrose. This isolates stained for 24 h with 1% coomassie blue stored in dionized sterile water and NAS R 250, 45% methanol, 45% water and slant tubes at 4°C for next uses. All 10% Acetic acid. After 5 h destaining strains compared with reference strain with 10% Acetic acid, gel were immersed IBSBF 669T of B. nigrifluens in in water, photographed, air dried for 24 h identification tests. and kept (Ausubel et al., 1987). Biochemical, physiological and Rep-PCR. Bacterial isolates were nutritional tests. All isolates were tested subjected to rep-PCR analysis using the as fallows: Solubility of bacterial cells in primers BOX AIR according to the 3% KOH (Suslow et al., 1982), gram procedure of (Versalovic et al., 1991, reaction (Schaad et al., 2001), oxidative 1994) in BIOMETRA termocycler. For (O) and/ or fermentative (F) glucose DNA preparation, bacterial cells grown metabolism (Hugh and Leifson, 1953), on Luria-Bertani broth in shaken culture oxidase (Kovacs, 1956), fluorescent at 27±1°C according to (Ausubel et al., pigment (Schaad et al., 2001), 1987). the amplified products were 59 A. JAMALZADE, M. SHAMSBAKHSH, H. RAHIMIAN electrophoretically separated in 1% spherical, white with smooth margin agarose gel at 5 V in TBE buffer (10.8 g on NA amended 5% sucrose after 2-3 tris, 5.5 g buric acid, 0.37 g EDTA in days at 27 ± 1°C. Some isolates in 1000 ml dionized sterile water) and first culture on modified EMB visualized with UV light after staining in produced purple color, but in ethidium bromide (0.5 μg/ml). The subculture produced green metallic molecular sizes of fragments generated sheen again, this isolates had further were estimated by comparison with simultaneously run Ladder Mix growing rate in compare to other (GeneRulerTM DNA Ladder Mix, isolates, but were not different in Fermentas). Lanes were compared by biochemical, physiological and reading horizontally across the gel image, nutritional properties. A few (less than from bottom to top; if a band was present, 15%) isolates were different in some it was assigned a value of 1 at that properties such as Levan production, location, whereas if absent, it was Esculin hydrolysis, H2S production assigned a value of 0. The from peptone, Arginine dehydrolase, presence/absence of bands was collated Nitrate reduction, Indol production into a binary data matrix. Cluster analysis and Methyl red reaction. These was performed on similarity matrices differentiations had not specific which were produced using similarity distribution and clustering of this coefficient and subjected to the between isolates on this basis was impossible.