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

Abstract

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 watersoaked 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 + . Key words: INA, vesicle protein, transmission electron microscopy ResumoLesões de mancha branca do milho causadas por Pantoea ananatis contribuiram substancialmente para a redução da produtividade nos cultivos de milho, em muitos países, incluindo o Brasil. Os sintomas iniciais da doença incluem lesões anasarcas nas folhas, que mais tarde tornam-se necróticas e de cor palha. O conhecimento básico sobre a biologia e os mecanismos de infecção deste patógeno são escassos. Neste estudo, 15 isolados de P. ananatis obtidos a partir de lesões da mancha branca do milho foram examinados quanto à sua atividade de nucleação de gelo (INA). A INA de isolados bacterianos individuais foi determinada por testes de nucleação em tubos. Isolados bacterianos foram cultivados em meio de caldo de soja tríptico e uma alíquota de 0,1 mL de cultura foi adicionada à tubos de ensaio contendo 1 mL de água destilada esterelizada. Os tubos foram colados em banho de gelo, o qual continha temperatura inferior a -10 ºC, por aproximadamente 2 min. A formação de gelo instantânea no tubo revelou um fenótipo INA positivo do isolado. Apenas 9 dos 15 isolados estudados apresentaram o fenótipo INA+. Testes de patogenicidade foram realizados com plantas inteiras e folhas destacadas.

Characterization of the inaA gene and expression of ice nucleation phenotype in Pantoea ananatis isolates from Maize White Spot disease

“…A comparison of these results with those reported in the literature confirmed that phenotype expression is determined by the environmental and physiological factors such as the age of bacterial culture, temperature, composition of the medium during growth, and plant and bacterial genotypes, in addition to the presence of the inaA gene in the cell (Lindow et al, 1978b;Nejad et al, 2006b;Sauer et al, 2014).…”

“…The INA phenotype is dependent on external factors, such as the age of the bacterium and culture conditions (Lindow et al, 1978b;Wysmierski et al, 2005;Nejad et al, 2006b;Sauer et al, 2014). Another study has also reported a relationship between the number of bacterial cells and ice-nucleation activity (Nejad et al, 2006a).…”

“…As the size of INA bacterial populations varies among different plant species, sites, climates, and seasons, only a small percentage of cells in a population are effective as ice nucleators (Pearce, 2001). The presence of glycerol in the culture medium is crucial to increase the number of external vesicles in the cellular wall of P. ananatis (Sauer et al, 2014). These vesicles contain INP, which are responsible for the formation of ice nuclei (Phelps et al, 1986;Kawahara, 2002).…”

“…In a recent study (Sauer et al, 2014), nine of fifteen P. ananatis isolates from MWS lesions from two different locations showed phenotypic variability for INA + . However, the presence or absence of the inaA gene in those isolates was not tested using molecular biological methods.…”

“…According to Lindow et al (1982a,b) and Lindow (1983), the INA + phenotype may be related to the virulence of P. ananatis. The ice formed by the bacterium in the intercellular spaces collapses the vegetal cell, leading to leakage of the cell contents, which in turn causes necrotic foliar spots, which are typical of MWS lesions (Lindow et al, 1982b;Sauer et al, 2014).…”