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Deciphering of Root and Rhizosphere microbiome to increase host fitness in the Fusarium oxysporum- plant interaction

Periodic Reporting for period 1 - DIRECTION (Deciphering of Root and Rhizosphere microbiome to increase host fitness in the Fusarium oxysporum- plant interaction)

Reporting period: 2018-09-01 to 2020-08-31

Fungal pathogens have a dramatic impact on crop productivity. Many fungal-plant interactions occur in the soil and crucially affect plant health. A particularly destructive group of root-infecting fungi cause vascular-wilts, which attack several crops, colonizing the roots and cause massive wilting. These diseases are amongst the most difficult to control, as these pathogens are usually inaccessible to chemicals being inside vasculature. Therefore, they are controlled with unspecific fumigation compounds that have a detrimental effect on soil quality.
Recently the impact of soil and plant associated microbiota on plant health has been widely studied. Plant associated Microbiome can be helpful for its development in many ways. One of the crucial roles this microbiota has, is to protect the plant from invading pathogens. It has been previously reported that beneficial microbes that associate with plants interfere with disease progression. However, very little is known about the interaction between members of plant associated microbiome and pathogenic fungi that shape the disease. Therefore, it is very important to understand how microbial interactions impact on shaping a plant disease outcome. This could be a sustainable solution against the plant vascular diseases.

The pathogenic fungus Fusarium oxysporum f. sp. lycopersici is a soil-borne pathogen which causes vascular wilt of tomato. The disease is difficult to control due to its non-symptomatic presence during the initial stages of fungal progression in plants. Currently control methods use extensive chemical pesticides which causes environmental pollution. As most soil fungicides are now banned by EU legislation, there is an urgent need to develop new control strategies, which need to be efficient, durable and environmental friendly. Identifying the microbial competitors or antagonists that interfere with Fusarium colonization could serve to protect the plants against this deadly disease. We hypothesized that native plant microbiome might have crucial role in controlling the disease progression of Fusarium. To investigate this, we defined the complete native microbiota from Tomato and have identified key antagonists which interfere with F. oxysporum growth. We further aim to re-construct an artificial synthetic microbial community (SynCom) by using there antagonists which could prevent the disease progression by F. oxysporum.
So, overall in terms of conclusions of the action we have defined the following:
1) A comprehensive native compartment specific microbiome of tomato with a collection of bacterial isolates.
2) Identified antagonistic bacteria that interact directly/indirectly with F. oxysporum during tomato colonization.
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).
Findings from my MSCA project, DIRECTION have resulted in new knowledge on how microbial interactions determine plant disease outcome. The identification of core microbiome in tomato plants that likely has an ability to interfere with colonization of a pathogenic fungus in planta. This will enable us to generate the more sustainable ways to control deadly pathogens like Fusarium for which limited sources of genetic resistance exists due to the developmental lethality. In addition, identifying the mechanistic basis of these interactions will provide new avenues to understand the re-constitution SynCom microbiome by using synthetic biology approaches. Overall, I believe results from my project will have broad implications, as they provide first glimpses of the crucial asymptomatic infection phases of these important pathogens that affect agronomically important crops.
My participation in conferences has allowed me to expand my professional network increasing my opportunities to become a successful Principal Investigator in the plant-microbe interaction field.
Plant root associated microbiome.