Investigation on the effects of biofector (biostimulants such as micro-organisms, algae extracts, mineral fertilizers such as rock P) applications on crop plant production is the overall aim of the project.

For Wageningen Plant Research, the specific aim is to investigate the impact of biofector applications on the endophyte community compositions of crop plants. It is believed that biofector applications can influence plant performances in agricultural settings by changing endophyte community compositions, because endophytes play important roles in resilience against plant diseases and stresses and can stimulate plant growth.

To test our hypothesis, we restrict ourselves to four biofector strains (Pseudomonas jessenii RU47, Bacillus amyloliquefaciens FZB42, Pseudomonas sp. Proradix strain, and Trichoderma harzianum, Trianum P) and two crop plant species (tomato and maize; i.e. plant models chosen by the consortium). We collected samples from plants treated with these biofectors (using untreated plants as controls) that were grown under different circumstances, to investigate impact of these biofectors on endophyte community composition. These circumstances were: 1) growth in pots filled with Hohenheim soil (low in P) in greenhouse facilities at JKI, Braunschweig, Ge, 2) plants treated with B. amyloliquefaciens grown in pots with potting soil in greenhouse facilities in Wageningen, NL, and 3) plants grown in the open field in Timisoara, Ru. The ecological behaviour of the applied biofectors in both plant species was investigated by us, and by other laboratories involved in the EU Biofector project. Surface-sterilized and peeled stem base samples from all tomato and maize plants were stored to up to four years at -70°C prior to DNA extraction.

For endophyte community analysis we will apply high throughput amplicon sequencing, using bacterial PCR primers directing the V3-V4 regions of the 16S rRNA gene. We spent much efforts to eliminate plant cell organelle amplicons from our samples because all plant DNA extracts contain high loads of plant cell organelles of prokaryotic origin such as mitochondria and chloroplasts. For that purpose, we evaluated different specific primer sets (based on primer 799, specifically designed to omit chloroplast amplifications), chloroplast-specific amplicon blockers and combinations of both before further downstream bacterial endophyte amplicon sequencing. Best-working method, in the sense that highest diversity of bacterial OTUs was obtained after amplicon sequencing, appeared to be the application chloroplast amplicon blockers. These blockers will be further used in all our analyses by high throughput amplicon sequencing.

        We planned an amplicon sequencing run on the Miseq platform for 2017. This work is still in progress. Data derived from the sequence run will be used for an experimental publication in an international peer-reviewed journal. Further, it is our intention to write a review paper on plant-associated microbial community shifts by plant applications with microbials. The EU-biofector project will end in October 2017.