ویژگی‏های فنوتیپی، بیماری‏زایی و مولکولی Xanthomonas arboricola pv. juglandis در استان‏های گرگان و مازندران

Xanthomonas arboricola pv. juglandis is the causal agent of walnut bacterial blight, the major nut and foliage disease of Persian walnut. Disease symptoms appear as necrotic lesions surrounded by chlorotic haloes on upper and lower leaf surfaces, stems, and apical necrosis on nuts. Affected aerial parts of walnut trees with suspected bacterial diseases symptoms were collected from all of the surveyed areas, and brought to the laboratory for further analysis. The samples were surface sterilized with dipping in 0.5 % sodium hypochlorite solution, washed twice with sterile distilled water (SDW) and dried on the paper tissue. A piece of each sample was cut using a sterile scalpel and macerated in 10-20 ml of SDW using a sterile mortar and pestle. A loopful of the resulting suspensions was streaked onto yeast-extract peptone glucose agar (YPGA) medium. The plates were incubated at 25-27°C for 48-72 h, and the resulted colonies were purified by repetitive streaking on fresh YPGA plates. Pale-yellow, domed circular, and convex colonies with entire margins of 1-2 mm in diameter on yeast extract-dextrose-calcium carbonate (YDC) agar medium were appeared. All the obtained bacterial isolates were subjected to the standard biochemical tests. All the bacterial isolates were positive for oxidative metabolism; levan production, catalase; hydrolysis of tween 80, gelatin, casein, aesculin and starch; as well as HR on tobacco leaves. On the other hand, the bacterial isolates were negaetive for oxidase and urease tests. Furthermore, bacterial isolates grow only on 1% and 3% NaCl. According to the phenotypic characteristics, walnut strains were identified as Xanthomonas sp. The isolates were further identified using generic and specific primers for Xanthomonads. The generic primer Xc-lip-F2/Xc-lip-R2 capable to amplify a 777 bp fragment in all isolates, and confirmed the bacterial isolates as Xanthomonas sp. The primer pair XarbQ-F/XarbQ-R specific for X. arboricola detected all isolates from walnut trees and the expected 402 bp fragment were amplified. The partial atpD, efp, gyrB and rpoD genes of bacterial strains were amplified, sequenced, and phylogenetically analyzed with other strains of the X. arboricola pathovars. The results of phylogenetic studies in X. arboricola strains isolated from walnut trees confirmed the identity of these strains as Xanthomonas arboricola pv. juglandis. Pathogenicity tests were conducted on walnut sapling in greenhouse conditions, and SDW was used as negative control. All of the isolates, produced the symptoms of disease on plants that artificially inoculated. Brown, necrotic spots, surrounded by a yellowish halo on leaves after 20 days of the artificial inoculation were observed, while the control plants inoculated with SDW remained healthy. Bacterial isolates, as those originally inoculated, were consistently re-isolated from artificially inoculated symptomatic plants on YPGA medium and their identity was confirmed by using PCR primers as described above.