Cultivation of Phytophthora palmivora, Colletrotrichum trifolii, and Rhizophagus irregularis and Inoculation of Plants

Fluorescently labeled P. palmivora LILI-td (accession {"type":"entrez-protein","attrs":{"text":"P16830","term_id":"137003","term_text":"P16830"}}P16830; Rey et al., 2015) expressing pTOR::tdTomato were cultivated on V8 vegetable juice agar plates as previously reported (Chaparro-Garcia et al., 2011). Seven-day-old plates of P. palmivora were flooded with 4°C sterile water and were incubated in the light at room temperature for 1 h to induce release of zoospores. For infection assays, suspensions of 5 × 10⁴ spores per mL were used. Roots of 7-d-old Medicago seedlings grown on square plates containing 0.8% (w/v) agarose were inoculated with 50 μL of zoospore suspension per plant.

C. trifolii was cultivated on OMA plates (Werner et al., 2007) and grown at 23°C under UV light for sporulation. After 14 d of growth, C. trifolii spores were washed from the plates using 0.02% (v/v) Tween20 and collected in Eppendorf tubes. Of the collected spores, 10⁵ were used for inoculating the roots of 7-d-old Medicago seedlings.

R. irregularis DAOM197198 was cultivated with Agrobacterium rhizogenes-transformed carrot roots in a bicompartment petri plate system as described (St-Arnaud et al., 1996). After 6–10 months of growth, spores were collected by dissolving agar pieces in 0.01 m citrate buffer (pH 6) at room temperature and with slow shaking in the dark overnight. Buffer was removed and spores were washed 2–3 times with water. Equal amounts of spores were used for plant inoculation.