Title: Immunofluorescence localization and ultrastructure of Stewart’s wilt disease bacterium Pantoea stewartii in maize leaves and in its flea beetle vector Chaetocnema pulicaria (Coleoptera: Chrysomelidae) Authors
Submitted to: Journal of Microscopy and Ultrastructure
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: January 27, 2014
Publication Date: May 17, 2014
Citation: Ammar, E., Correa, V.R., Hogenhout, S.A., Redinbaugh, M.G. 2014. Immunofluorescence localization and ultrastructure of Stewart’s wilt disease bacterium Pantoea stewartii in maize leaves and in its flea beetle vector Chaetocnema pulicaria (Coleoptera: Chrysomelidae). Journal of Microscopy and Ultrastructure. 2:28-33. Interpretive Summary: Stewart's wilt of corn is an important disease of sweet corn in the U.S. Although grain corn is less affected by the disease, concerns about seed transmission of Stewart's wilt can restrict exportation of corn from parts of the Corn Belt where the disease occurs. Stewart's wilt is caused by a bacterium called Pantoea stewartii. This bacterium is transmitted between plants by the corn flea beetle, and it is generally thought that the bacterium overwinters in the gut of this insect. In a previous study, we identified a set of genes in P. stewartii that are required for the bacteria to persist in the insect gut. Because the genes we found encode a structure that bacteria use to invade the cells and tissues of hosts, we hypothesized that the bacteria may have a more intimate relationship with host cells, and not simply adhere to the gut surface. We used microscopic techniques to determine where the bacteria were in both corn and the flea beetle. As expected, we found P. stewartii mainly in the xylem and intracellular spaces of infected corn leaves. Similarly to our previous experiments, we found the bacteria in the insects gut for up to 12 days after feeding on infected plants. When we examined the distribution of bacteria in the insect gut using electron microscopy, we found cells similar to P. stewartii in the gut cavity close to the microvilli, the cells that absorb nutrients in the gut. Importantly, we also saw the bacterial cells inside cells in the insect's midgut, suggesting that P. stewartii does indeed infect insect cells. These results will provide the basis for further research to determine the role of intracellular persistence of the bacteria in the spread of disease that may provide new targets for controlling Stewart's wilt of corn.
Technical Abstract: Pantoea stewartii is the causal agent of Stewart's wilt of sweet corn, the most serious bacterial disease of sweet corn and maize in the North-Central and Eastern USA. P. stewartii is transmitted mainly by the corn flea beetle Chaetocnema pulicaria (Coleoptera: Chrysomelidae) and this bacterium is assumed to overwinter in its vector beetle. Using immunofluorescence confocal and transmission electron microscopy (TEM) we localized P. stewartii in its host plant and insect vector. In infected maize, P. stewartii was found mainly in xylem vessels of major and minor veins as well as in intercellular spaces of infected leaves. In the vector beetle, P. stewartii was found by immunofluorescence in the foregut, midgut and hindgut up to 12 days post-acquisition (post-feeding on infected plants for 2 days). TEM of thin sections in the beetle's gut four and eight days postacquisition revealed bacterial cells similar to those of P. stewartii in the gut lumen, close to or associated with the gut microvilli, as well as inside epithelial cells of the midgut. This suggests a stronger biological relationship between this bacterium and its vector, and that the corn flea beetle may carry P. stewartii intracellularly as well as extracellularly within the gut, which has implications in the persistence and overwintering of this bacterium in the vector and on the epidemiology of Stewart's wilt disease of corn.