Dmlb tilting trinocular compound microscope

A range of compounds which interfere with Ca²⁺ cell signaling via different mechanisms were chosen and tested at various concentrations for their impact on S. subterranea zoospore motility. These compounds included lanthanum (III) chloride (LaCl₃) and gadolinium (III) chloride (GdCl₃), ethylene glycol-bis (β-aminoethyl ether)-N,N,N’,N’-tetraacetic acid (EGTA), trifluoperazine (TFP), and amiloride hydrochloride. Various concentrations of these compounds were tested for their effects on zoospore flagella movement. One microliter zoospore suspension was added to 5 μl of each test compound at 50–250 μM in a taxis chamber, mixed well and allowed 10 min to acclimatize before observing for zoospore flagella movement at 400 × using light microscope (Leica DMLB tilting trinocular compound microscope, Leica Microsystems, Wetzlar, Germany). This was repeated for TFP at 5–25 μM, and EGTA at 1,000–2,000 μM. Each trial was replicated five times. Chemical doses that caused a cessation of flagella movement were classified as lethal and excluded from subsequent trials.

Root tissues were excised from 6-week-old axenic tissue-cultured plantlets of potato cv. Iwa, a variety highly susceptible to S. subterranea root infection. Roots were triple rinsed in sterile deionized water, cut into c. 1 cm long pieces and placed two apiece onto a microscope slide. Each slide was then flooded with 45 μl of the various Ca²⁺ antagonist solutions followed by 15 μl of zoospore suspension (c. 11 zoospores/μl) added to the outer margins of the antagonist solution to prevent direct contact of the zoospore suspension with the root pieces and a cover slip carefully added. To prevent the drying, the prepared slide was placed on moistened Whatman filter paper (GE Healthcare, Chalfont Saint Giles, United Kingdom) within a Petri dish, to create high humidity in the chamber. Each Petri dish constituted a replicate with each treatment replicated six times. The set up was incubated for 12 h in a dark cabinet at room temperature (20 ± 2°C). Following incubation, the root pieces were carefully removed with forceps, gently rinsed in sterile deionized water to remove unattached zoospores, mounted on a microscope slide, and observed by light microscopy at 400 × magnification (Leica DMLB tilting trinocular compound microscope, Leica Microsystems, Wetzlar, Germany). The number of zoospores attached to the root and root hairs were counted.