Monitoring the establishment of a synthetic microbial community with a potential biocontrol activity against grapevine downy mildew using a microfluidic qPCR chip

Abstract

Grapevine downy mildew, caused by the oomycete Plasmopara viticola, is responsible for significant economic losses each year and for a large proportion of the fungicides used in viticulture. In order to limit the use of these chemical pesticides, which are incompatible with the development of sustainable viticulture, biocontrol solutions based on cultivated simplified communities of microorganisms (SimComs) are gradually emerging.

In the present study, we designed several SimComs for the control of downy mildew, using a collection of microorganisms isolated from grapevine leaves by a culturomic approach. The SimComs are composed of bacteria, yeasts and filamentous fungi, described to have either a biocontrol activity against plant pathogens or abundant on grapevine leaves. We tested the hypothesis that including abundant species in the SimComs would help the microbial community colonize the leaves.

A quantitative PCR microfluidic chip (Fluidigm Biomark) was developed to monitor the establishment of SinComs on grapevine leaves. Larger number of reaction are allowed by microfluidic PCR compared to classical qPCR, resulting in quicker and cheaper price per sample. It also has the advantage of providing absolute abundance data compared to metabarcoding approaches that only estimate the relative abundance of microbial taxa.

So far, specific primers for 34 microbial taxa (out of the 42 selected for inclusion in the SimComs) have been designed in single-copy housekeeping genes. We are currently sequencing the genomes of the remaining microbial taxa to complete the primer design. We applied the microfluidic chip to DNA samples extracted from grapevine leaf discs inoculated with SimComs and were able to detect most of the inoculated microorganisms, including some microbial taxa that significantly reduced the intensity of downy mildew symptoms under laboratory conditions.

The microfluidic chip was then applied to environmental DNA collected in vineyard from spore sensor, in order to detect and quantify the targeted protective microorganisms. By doing so, we were able to detect the presence of several microorganisms, including some microbial taxa with proven biocontrol activity against plant pathogens such as A. pullulans and E. nigrum.

These preliminary results shed light on the potential of microfluidic chips as a new molecular diagnostic tool to monitor specific microbial communities present naturally or artificially after Simcom inoculation in the field. Grapevine downy mildew, caused by the oomycete Plasmopara viticola, is responsible for significant economic losses each year and for a large proportion of the fungicides used in viticulture. In order to limit the use of these chemical pesticides, which are incompatible with the development of sustainable viticulture, biocontrol solutions based on cultivated simplified communities of microorganisms (SimComs) are gradually emerging.

In the present study, we designed several SimComs for the control of downy mildew, using a collection of microorganisms isolated from grapevine leaves by a culturomic approach. The SimComs are composed of bacteria, yeasts and filamentous fungi, described to have either a biocontrol activity against plant pathogens or abundant on grapevine leaves. We tested the hypothesis that including abundant species in the SimComs would help the microbial community colonize the leaves.

A quantitative PCR microfluidic chip (Fluidigm Biomark) was developed to monitor the establishment of SinComs on grapevine leaves. Larger number of reaction are allowed by microfluidic PCR compared to classical qPCR, resulting in quicker and cheaper price per sample. It also has the advantage of providing absolute abundance data compared to metabarcoding approaches that only estimate the relative abundance of microbial taxa.

So far, specific primers for 34 microbial taxa (out of the 42 selected for inclusion in the SimComs) have been designed in single-copy housekeeping genes. We are currently sequencing the genomes of the remaining microbial taxa to complete the primer design. We applied the microfluidic chip to DNA samples extracted from grapevine leaf discs inoculated with SimComs and were able to detect most of the inoculated microorganisms, including some microbial taxa that significantly reduced the intensity of downy mildew symptoms under laboratory conditions.

The microfluidic chip was then applied to environmental DNA collected in vineyard from spore sensor, in order to detect and quantify the targeted protective microorganisms. By doing so, we were able to detect the presence of several microorganisms, including some microbial taxa with proven biocontrol activity against plant pathogens such as A. pullulans and E. nigrum.

These preliminary results shed light on the potential of microfluidic chips as a new molecular diagnostic tool to monitor specific microbial communities present naturally or artificially after Simcom inoculation in the field.