Detection and Identification of Erwinia carotovora Subsp. Atroseptica ( Van Hall , 1902 ) the Causal Agent of Potato Blackleg by RFLP-PCR

Potato blackleg disease is surveyed for spreading out in different potato growing areas in Jordan including; Jordan Valley (Al-Karama, Ashshuna Al-Janubiyya, Muthallath Al-Misri, Mu’addi, Deir Alla, Dirar, and Kurayyima), Amman (Al-Yadoda), Madaba (Om Al-Amad), Jerash (Tawahin Al-Udwan), Ma’an (Rum, and Al-Mudawwara). Forty six bacterial isolates of Erwinia carotovora subsp. atroseptica (Eca), the causal agent of the disease, were isolated and identified by different biochemical, physiological and nutritional tests from 145 diseased potato samples collected. Polymerase chain reaction (PCR)-based amplification of a 690-bp band was used to confirm the presence of Eca in the isolated bacteria estimated by the reference culture (ATCC 15713). PCR amplification was followed in isolation, identification and detection of Eca in infected and symptomless potato-stem and- tuber samples. The results showed that Eca was isolated from 30% of naturally infected potato samples with blackleg, while the 690-bp band was detected in 88% of DNA extracts from the naturally infected potato samples. In the other hand Eca was not isolated from the symptomless potato stem samples, while the 690-bp band was detected in 8% of the DNA extract from these stem samples. However; using symptomless-tuber samples have improved isolation efficiency. Eca was isolated directly from 4% of the samples when their suspension was streaked on Logan’s medium plates however 690-bp band was detected from 32% of their DNA-extract amplified with PCR. Genetic variability among the Eca isolates has been able to detect 690-bp PCR products for 46 different isolates those were digested with the restriction enzymes i.e. AluI, HaeII, HpaII and SauAI. Polymerase chain reaction-Restriction fragment length polymorphism (PCR-RFLP) analyses were able to cluster the Eca isolates into five PCR-RFLP groups with low genetic variability.