In my MSCA fellowship, DIRECTION, I defined the native microbiota of Tomato from soil as well as different root compartments. For the purpose I followed the culture independent and culture dependent approaches for microbiome analysis. The compartments selected for analysis included soil, rhizosphere soil, whole root and endophytic root. Considering that the F. oxysporum colonizes xylem as its ultimate destination, we also defined bacterial communities from xylem sap in order to define key microbial antagonists.
The culture dependent method led us to isolate around 800 bacterial strains from all compartments to form culture collection. By a complementary cuture independent analysis, we identified core dominant phyla present in all root compartments. Comparatively the members of dominant phyla found to be present in our culture collection. Next, we used the representative strains of all dominant phyla to carry out a high throughput interaction screening between F. oxysporum and individual bacterial isolate. I have standardized this protocol for high throughput screening by using GFP reporter strain of F. oxysporum to see the direct/indirect effect of the bacterial isolates on F. oxysporum growth by measuring the GFP fluorescence. This assay allowed us to select antagonistic bacterial strains against Fusarium in-vivo. These selected antagonists from fluorescence assay were further used for investigating the mechanistic basis of this microbe-microbe interaction and how they shape vascular wilt disease.
The changes in bacterial community dynamics was investigated upon inoculation of Fusarium by different methods which included F. oxysporum inoculation in either soil or vermiculite that mimic the agricultural setting. Moreover, we also used a dipping inoculation method which is routinely used for in vitro pathogenicity assays to investigate changes in bacterial community dynamics. These different methods have shown the differentials in community changes which determine the changes in microbiome with Effect of these selected antagonists were investigated by also performed the analysis to understand the impact of inoculum and the nature of infection method on microbial interactions.
We also investigated the interference of these selected bacterial antagonists on F. oxysporum growth in planta on tomato roots under semi sterile conditions. These experiments accelerated the selection of bacterial antagonists that show an effect on F. oxysporum by either showing a drastic decrease in mortality which resulted from inoculation of fungus alone in absence of the bacterial antagonists or an intermediate phenotype which resulted in decrease of the disease index of F. oxysporum mortality on tomato seedlings.
Current experiments aim to understand the tripartite interaction between tomato host, antagonists’ bacteria and F. oxysporum by using Electron microscopy to understand the ultrastructural changes of physiological relevance that determine the outcome of these interactions.
Moving forward, I will continue to advance findings from my MSCA project in my host lab through the Postdoctoral Fellowship I have won from the University of Cordoba. I aim to dissect the functional characterization of this identified microbial interaction. This work is expected to result in two research manuscripts detailing my findings which are currently in final stages of their preparation. In addition to the project work, I was also the first author of one commentary article: which describes how the effectors from filamentous plant pathogens are mimicking the components of host immunity (
https://doi.org/10.1016/j.tplants.2019.06.009(se abrirá en una nueva ventana)).
