Evaluation of Ice Nucleation Activity (INA) and INA Gene Detection in the Bacteria Isolated from Pistachio Trees in Kerman Province, Iran

IIce nucleation active (INA) bacteria are common epiphytic inhabitants that cause frost damage in many plants in the near-zero temperatures. Yet, no studies were found in ice nucleation bacteria associated with pistachio trees. In our earlier study some INA strains were identified and reported. These were assigned as Pseudomonas fragi, P. putida, P. moraviensis and Pantoea agglomerans. In current work, two new strains namely P. viridiflava and Entrobacter cloacea were identified. Their ice nucleation frequency were evaluated and compared with above-mentioned ice positive strains isolated from pistachio trees. Pseudomonas fragi raf3 was considered as the most ice nucleation active bacteria. This was followed by P. putida raf6, P. moraviensis raf1, P. moraviensis raf5, Pantoea agglomeranse raf7, P. viridiflava raf2, Entrobacter cloacea raf8 and Pseudomonas sp. raf4, respectively. To detect INA genes, two sets of degenerate primers were used and partial INA gene sequences were amplified. INA gene sequence) 425bp) for Pseudomonas putida raf6,  Pantoea agglomerans raf7 and P. fragi raf3 were amplified with primer pair of 3308/3463. Whereas, a fragment of 194bp was detected in Pseudomonas sp. raf4,  P. moraviensis raf5 and  P. moraviensis raf1using forward and reverse primer pair of 3076/3463. Entrobacter cloacea raf8 has reported for the first time as epiphytic ice plus strain. The capability of the latter as a bacterial biocontrol agent against insect pests was reported.