Various bacteria belonging to Enterobacteriaceae are known to be agents causing blackleg and soft rot in potato. In this study, a rapid and specific detection method was used to investigate bacteria in some northwestern provinces of Iran (Zanjan, Kordestan and eastern Azarbaijan). 26 strains were selected for the study that had been identified in previous studies as representative pathogens, they were as follows: 14 Pectobactrium carotovorum subsp carotovorum, 7 Dickeya chrysanthemi and 5 Pectobactrium atrosepticum. Primers Y1/Y2, Expccf/r, ADE1/2 and Eca1f/r, published as specific primers for the pathogens. They were tested to determine if they could be used for specific amplification of DNA in the strains. The expected specific fragment was not amplified in many strains or several non-specific bands, some close to those expected fragments were amplified. Specific primers for amplification of the DNA of pathogens were designed by sequencing the ITS region from representative strains and a PCR test was developed. In the PCR test a 300 bp fragment was amplified from all strains collected from the provinces. In specificity tests, no PCR products were obtained from other bacteria belonging to other genera.
In vitro screening techniques were used to evaluate 46 genotypes of Iranian potato collection for resistance to bacterial soft rot caused by Pectobacterium carotovorum subsp. carotovorum (Pcc). One month old in vitro rooted potato plantlets were inoculated by two inoculation techniques under in vitro conditions: 1) sterile toothpicks dipped into bacterial suspension and pressed into the crown of plantlets and 2) the freshly cut crown of plantlets were dipped into bacterial suspension of 108 cfu ∙ ml-1 for 10 min. Typical soft rot disease symptoms, including the percentage of wilted leaves were recorded on inoculated plantlets 3, 6, 9, 12 and 15 days post-inoculation. The potato genotypes which were examined responded differently to Pcc and varying levels of resistance were observed. Potato genotype AG showed the highest level of resistance. Results obtained from in vitro screening were then verified by classical tuber slice assay. The verifications were conducted using five representative cultivars: Milva, Ramus, Picaso, Marfona and Agria which responded similarly to both in vitro and classical evaluation systems. Similar results obtained from these tests indicated that the in vitro screening technique developed in this study could provide a simple and rapid whole plant assay in selecting resistant potato genotypes against bacterial soft rot.
To find the best inoculation method for evaluation of the resistance in potato genotypes against bacterial blackleg caused by Pectobacterium atrosepticum under in vitro conditions, five inoculation methods were compared. In vitro grown explants of five potato genotypes were inoculated with different inoculation methods, then placed on MS solid medium and incubated at 23?C with 70% relative humidity under the light regime of 16 hours a day. After the appearance of symptoms, the efficiency of inoculation methods was then recorded based on the severity of disease symptoms in potato genotypes: Farmosa, Agria, Picaso, Marfona and a wild potato genotype ?Solanum phureja'. Plantlets inoculated by piercing the crown with sterile toothpick inoculated in bacterial suspension of 108 cfu/ml showed the most severe symptoms. Based on all experiments, cultivar Marfona showed higher resistance among all cultivars and, cultivar Agria was the most susceptible. Finally, after witnessing the reactions of different varieties to inoculation methods and comparing them with previous evaluations of resistance in greenhouse conditions, the crown treatment employing sterile toothpick after infection in 108 cfu/ml bacterial suspension was selected and introduced as the best evaluation method of in vitro potato explants against blackleg.
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