تعیین فیلوتیپ و سکووار جدایه های باکتری Ralstonia solanacearum، عامل بیماری پژمردگی باکتریایی سیب زمینی در جنوب استان کرمان

The southern Kerman province (Jiroft) is one of the main regions for potato production in Iran. Bacterial wilt is a serious disease of potato in this region. The pathogen, Ralstonia solanacearum species complex, is divided to 4 phylotypes and 57 sequevars. In May 2017, potato fields of the Jiroft region were surveyed. Tuber samples were collected from the diseased potato plants showing typical bacterial wilt symptoms. Pure bacterial cultures were isolated from the tubers on Kelman's triphenyltetrazolium chloride agar medium and a total of 21 isolates were obtained. The R. solanacearum species complex-specific primer pair 759/760 was used to confirm the identity of all the isolates. The biovar of the isolates was determined on the basis of the utilization of disaccharides and hexose alcohols. Furthermore, the phylotype assignment of the isolates was carried out by a Pmx-PCR (phylotype multiplex PCR) with primers targeting the internal transcribed spacer region (four forward primers each specific for one phylotype and one reverse primer). To determine sequevar of the isolates, a 750-bp DNA fragment from endoglucanase (egl) gene was amplified from the genomic DNA of each isolate by the primer pair Endo-F and Endo-R. PCR products were sequenced and a phylogenetic tree was constructed by MEGA 6.0 software using the neighbor-joining method. Results of biochemical tests indicated that all isolates belong to the biovar 2. Moreover, in Pmx-PCR test, two DNA fragments with the size of 372 (the phylotype specific amplicon) and 281bp (R. solanacearum-specific 281 bp amplicon) were amplified from the isolates genomic DNA. The phylogenetic analysis of the egl gene sequences of the R. solanacearum isolates and comparison of the obtained sequences with those available in Genbank and Pmx-PCR revealed that all of the tested isolates belong to phylotype IIB and sequevar 1. The sequevar and phylotype identification of the isolates were also confirmed by R. solanacearum typing program software. This is the first study to report the determination of phylotype and sequevar of R. solanacearum isolates infecting potato in Kerman province.