Ice nucleation activity in Pantoea ananatis obtained from maize white spot lesions

Maize white spot lesions caused by Pantoea ananatis has contributed substantially to yield reduction of maize crops in many countries, including Brazil. The initial symptoms of the disease include water-soaked lesions on the leaves, which later become necrotic and straw-colored. Basic knowledge regarding the biology and the infection mechanisms of this pathogen is lacking. In this study, 15 P. ananatis isolates obtained from maize white spot lesions were examined for their ice nucleation activity (INA). The INAs of individual bacterial isolates was determined by tube nucleation tests. Bacterial isolates were grown on tryptic soy broth medium and an aliquot of 0.1 mL of culture was added to test tubes containing 1 mL of sterile distilled water. The tubes were packed in an ice bath, which had a temperature below –10°C, for approximately 2 min. Instantaneous formation of ice in the tube revealed a positive INA phenotype of the isolate. Only 9 of the 15 studied isolates showed the INA+ phenotype. Pathogenicity tests were performed using whole plants and detached leaves. Symptoms were reproduced in both tests, but only for the inoculations using INA+ isolates. Electron microscopy allowed visualization of protein vesicles under outer cell wall of isolates characterized as INA+.