ORIGINAL ARTICLE Genetic diversity of Iranian Agrobacterium strains from grapevine Kiomars Rouhrazi & Heshmatolah Rahimian Received: 18 November 2011 / Accepted: 25 January 2012 / Published online: 29 February 2012 # Springer-Verlag and the University of Milan 2012 Abstract Crown gall of grapevine is an economically im- portant disease in all areas where grapes are grown, but it is particularly severe in regions with cold climates. The pur- pose of this study was twofold: (1) to compare available Agrobacterium primer sets and identify a more reliable PCR analysis that can detect tumorigenic and non-pathogenic A. vitis and A. tumefaciens strains involved in the crown gall disease of grapevines in Iran and (2) to detect the genetic diversity of these strains by insertion sequence 50 (IS50) PCR as the genetic marker. Based on the results of morpho- logical, physiological, biochemical, and pathogenicity tests and amplification of the virD2 gene fragment and subse- quent comparison of IS50-PCR fingerprints of the strains, we identified 27 of all 40 strains tested as tumorigenic A. vitis, three strains as non-tumorigenic A. vitis, and ten strains as tumorigenic A. tumefaciens. The similarity of all pairwise combinations was determined using Jaccard’ s coefficient, and cluster analysis was performed using the unweighted pair group method with arithmetic average (UPGMA) clus- tering. On the basis of the fingerprint patterns generated by IS50-PCR,, the isolated strains were differentiated into four clusters (A–D) at the 60% similarity level. These four clus- ters could be further divided into several subgroups at the 80% similarity level. These results demonstrate that there is a wide variation in the sensitivity patterns of different Agro- bacterium strains to different antibiotics. Keywords Agrobacterium . Genetic diversity . Grapevine . Antibiotic sensitivity Introduction Crown gall caused by tumorigenic Agrobacterium vitis and A. tumefaciens occurs in grape-growing areas worldwide (Burr et al. 1998). The crown gall bacterium was first isolated in the USA from Paris daisy by Smith and Townsend (1907). Infected vines can harbor both pathogenic and non-pathogenic strains and remain symptomless until the vines are injured (Burr and Katz 1983). There is no effective chemical control, and preventive measures, including early detection of the pathogen and propagation of disease-free planting material, are currently the only control measures available (Burr et al. 1998; Burr and Otten 1999). Phylogenetic analyses of tumorigenic and non- pathogenic A. vitis strains were carried out in Japan using the results of repetitive sequence-based PCR analyses and the partial nucleotide sequences of pyrG, recA, and rpoD (Kawaguchi et al. 2008). In subsequent study by the first author (Kawaguchi 2011)s, 41 tumorigenic and non- tumorigenic strains, respectively, of isolated from Japan were separated into seven clades using multilocus sequence analysis (MLSA) of the partial nucleotide sequences of pyrG, recA, and rpoD. The crown gall bacterium was first isolated in Iran in Qazvin province (Amani 1966). This disease has been reported in different regions of Iran, including Fars, Kohgi- luyeh and Boyer–Ahmad, Takestan, Karaj, Tehran, and Qaz- vin provinces (Ale–Yasin and Banihasemi 1993; Fatehi et al. 1998; Javaheri et al. 2000; Salahi-Ardakani et al. 2000; Salehi et al. 2006). To date, however, there has been no systematic collection, differentiation, and characterization of Agrobacte- rium strains isolated from Iran vineyards and naturally grow- ing wild grapes. In Iran, crown gall can lead to vine decline and mortality in vineyards. Grapevines grown in areas subject to freezing winter temperatures are especially vulnerable to K. Rouhrazi : H. Rahimian (*) Plant Bacteriology Laboratory, Department of Plant Protection, Sari Agricultural Science and Natural Resources University, Sari, Iran e-mail: Rahimian.H@gmail.com Ann Microbiol (2012) 62:1661–1667 DOI 10.1007/s13213-012-0423-x