Detection of viable urediniospores, morphological characterization in resistant and susceptible genotypes, and germ tube anastomosis of Phakopsora pachyrhizi.

摘要

Soybean rust, caused by Phakopsora pachyrhizi, is a major foliar disease of soybean affecting soybean yields in many soybean-growing areas throughout the world. After the reports of its first occurrence in Brazil in 2001 and the continental United States of America in 2004, research on the disease and its pathogen has greatly increased. The objectives of my study were to i) develop a multiplexed immunofluorescence assay to identify and detect viable P. pachyrhizi urediniospores, ii) characterize the infection and colonization of P. pachyrhizi on soybean genotypes with varying levels of resistance using microscopic observations and a quantitative-polymerase chain reaction assay, and iii) demonstrate germ tube anastomosis and nuclear migration during the germination of P. pachyrhizi urediniospores. I developed a rapid and reliable technique for the detection of viable P. pachyrhizi by integrating an immunofluorescence assay with propidium iodide staining. In this two-color fluorescence assay, live spores stained green and were distinguished from dead spores that also stained green but with their nuclei clearly stained red. The method has the potential to be used in soybean rust forecasting systems that depend on monitoring airborne urediniospores through capture by various trapping techniques and need to distinguish between live and dead urediniospores. In the second study, microscopic observations of the infection process in soybean genotypes distinguished complete (immunity), and incomplete resistance from each other and from susceptibility. Pre-penetration and penetration by P. pachyrhizi were similar among different genotypes and differences in the infection process were more evident once the hyphae penetrated into the intercellular spaces of the mesophyll. The susceptible cultivar Williams 82 had extensive hyphal growth in the mesophyll cells. The soybean cultivar UG5 conferred complete resistance, and plant introductions 567102B and 224268, which were classified as having incomplete resistance, had extensive necrosis of the mesophyll cells. These results, in combination with fungal DNA concentrations, are the first detailed evidence of quantitative differences in the infection process of P. pachyrhizi among soybean genotypes varying in levels of resistance. In the third study, I observed that fusion of germ tubes in germinating urediniospores of P. pachyrhizi resulted in a complex hyphal network. Nuclear staining showed their migration of through the network resulting in multinucleate hyphae and provided the first evidence of anastomosis in P. pachyrhizi. Considering the lack of a known sexual stage of P. pachyrhizi, hyphal anastomosis might explain the genetic diversity in virulence among populations of P. pachyrhizi..