Establishing methods, protocols for generating and using spores and protoplasts in C. austroafricana

In the control column, "+" indicated days after, while "-" indicates days before the control produced pycnidia.</p><p dir="ltr">Table 2.3 Calculated molar concentrations(M), solute potentials(Ψs), water potentials(Ψw), and water activities (aw) of the media used in the water potential trials at various concentrations of Sorbitol and KCl combined with 2% agar medium.</p><p dir="ltr">Table 2.4 Recordings of first observed pycnidia emergence for tested isolates with KCl (K) and sorbitol (S) as additives.

A column indicating the time difference with the control (in days) is also given. “+”indicates days after. “-“ indicates days before. “—” indicates that no pycnidia formed.</p><p dir="ltr">Table 2.5 First observed pycnidia emergence at various temperatures. A column indicating the time difference with the control (in days) is also given. "+" indicates days after, while "-" indicates days before the control. "—" indicates that no pycnidia formed.</p><p dir="ltr">Table 2.6 First observed pycnidia at different pH ranges on oatmea lagar(O) and malt extract agar(M).

A column indicating the time difference with the control (in days) is also given. "+" indicates days after, while "-" indicates days before the control. "—" indicates that no pycnidia formed.</p><p dir="ltr">Figure 3.1 Core steps of new methodology. A) Solid media plate with formed pycnidia is flooded with 2% NaCl and allowed to soak with periodic shaking/tapping. B) Suspended spores are collected.

Orange colour shows high concentration. C) Spores are centrifuged into a pellet, and the supernatant is removed. D) Pellets are resuspended into high concentration.</p><p dir="ltr">Figure 3.2 Ungerminated spores at 0 hr are shown (left).

Overmatured germlings (mycelia) are shown at 20 hrs (right). Photos captured using Countess 3 automatic cell counter with incubation occurring in 1% liquid malt extract. Zoom approximately X250.</p><p dir="ltr">Figure 3.3 Recently germinating germlings.

Photo taken at 18h40 using Countess 3 automatic cell counter. Zoom approximately X250.</p><p dir="ltr">Figure 3.4 Formation of protoplasts in 10% Extralyse with 1.2 M NaCl. Bar represents 10 µm.

A) Shown is a protoplast approximately 3 µm in size. Most protoplasts were this size. B) Shown is a protoplast approximately 6 µm in size forming from the hyphal tip of a mycelium.

C) Potential formation of a large 9 µm protoplast from a more mature hypha. Direct release was never observed, and such large protoplasts were rare, yet they were believed to be derived from such mature mycelia.</p><p dir="ltr">Figure 3.5 Photos taken from Trial C. The bar in both represents 10 µm. A) To the left is shown protoplasts in OC1 at 4h45.

B) To the right is shown large, oblong, and malformed protoplasts that formed in OC2 by 5 hours.</p><p dir="ltr">Figure 3.6 Protoplasts in both GOM (Left) and OC4(Right) were prone to remain associated with mycelia. Photos taken at 4h45 and 5 hours respectively.</p><p dir="ltr">Figure 3.7 A) GOM after being stored in 4°C for a week, observed after 6 hours of digestion. Hyphae appear intact and protoplasts are forming B) OC1 after being stored in 4°C for a week, observed after 6 hours of digestion.

Hyphae appear well digested. Swollen, large, and oblong protoplasts are seen forming. C) OC2 after being stored in 4°C for a week, observed after 6 hours of digestion.

Hyphae appear disintegrated. Swollen, large, and oblong protoplasts are forming. Some cells lack organelles.

D) OC3 after being stored in 4°C for a week, observed after 6 hours of digestion. Hyphae appear intact, and protoplasts are forming.</p><p dir="ltr">Figure 3.8 GOM results from trial D A) An interphase layer consisting entirely of protoplasts was formed (1) using GOM with the gradient isolation technique. B) Protoplasts re-isolated from the interphase layer at X600.

These were larger than often encountered with sizes ranging from 5 to 10 likely owing to swollen vacuoles.</p><p dir="ltr">Table 3.1 Lists the tested buffers, which are further defined in the appendix section under "Buffers", as well as conditions for each trial.</p><p dir="ltr">Trial outcomes consist of Tables 3.2, 3.3, and 3.4:</p><p dir="ltr">Table 3.2: Both 1.2 M NaCl and 1M Sorbitol were tested at various Extralyse™ concentrations of 0.1%, 1%, 5%, and 10%, respectively.

In all cases, protoplasts could not be visually confirmed. Failed trials were visualized on a Countess 3 automatic cell counter, which was later determined to lack the required magnification resolution to visualize protoplasts.</p><p dir="ltr">Table 3.3: Protoplast formation and stability under a 10% Extralyse™ concentration using different buffers. Various buffers were tested for their ability to sustain protoplast formation and stability under a 10% Extralyse™ concentration.</p><p dir="ltr">Table 3.4: Comparison of two protoplast isolation methods.

Gradient centrifugation versus filtration using Whatman No.4. Here, Whatman No.4 was found to remove protoplasts from samples.</p><p dir="ltr">Buffers: Contains information on buffer mixtures used during experimentation of protoplasts throughout the dissertation.</p><p dir="ltr">Equations: contains a summary of the equations used in Chapter 2 to standardise osmotic pressures between trials.</p>