FIGURE 2.

Exogenous application of Austropuccinia psidii‐specific double‐stranded RNA (dsRNA) decreases the incidence of myrtle rust (MR) on Syzygium jambos in detached‐leaf assays. S. jambos leaves (n = 4) infected with A. psidii and treated with 11 A. psidii‐specific dsRNAs were assessed 2 weeks postinoculation. Treatments consisted of 10⁶ A. psidii spores/ml in sterile distilled water containing 0.05% Tween 20 mixed with 100 ng/μl dsRNA. −dsRNA (control) was A. psidii spores in sterile distilled water and 0.05% Tween 20. Green fluorescent protein (GFP) dsRNA was included as a nonspecific control. (a) Boxplot with superimposed scatter representing the mean number of A. psidii pustules per S. jambos leaf (n = 3) treated with 11 A. psidii‐specific dsRNAs. Significance as compared to the control (−dsRNA) is represented by grey asterisks (*p < 0.05, **p < 0.01, ***p < 0.001; Student's t test). This figure was made in R v. 4.0.3 (R Core Team, 2020). (b) Photo comparisons of all dsRNA treatments and controls on S. jambos leaves. For each gene target a control (−) and treatment (+) leaf is shown. Each treatment leaf had a respective control leaf from the sample plant and of the same age, size, and growth stage to ensure accurate comparison. Colours from green to orange represent most to least significant reduction in spore number from control to treatment, respectively. All treatments to the left of the dotted line (ribosomal RNA 28S‐1 (28S‐1), β‐tubulin (β‐TUB), translation elongation factor 1‐α (EF1‐a), mitogen activated protein kinase (MAPK), acetyl CoA‐transferase (ATC), glycine cleavage system‐H (GCS‐H), cytochrome P450 (CYP450) and haustorial target (H01215)) resulted in a significant reduction in pustule number compared to control leaves. All treatments to the right of the dotted line (haustorial target H01136, ribosomal RNA 28S‐2 (28S‐2) and haustorial target H12890) did not result in a significant reduction in pustule number from control to treatment leaves. (c) Boxplot with superimposed scatter representing GFP (nonspecific control) mean number of A. psidii pustules per S. jambos leaf (n = 3) treated with 11 A. psidii‐specific dsRNAs. Significance as compared to the control (−dsRNA) is represented by grey asterisks (*p < 0.05, **p < 0.01, ***p < 0.001; Student's t test). This figure was made in R v. 4.0.3 (R Core Team, 2020). (d) Close‐up photo comparison of control (A. psidii only) and treatment (A. psidii + β‐TUB dsRNA) on S. jambos leaves, taken 2 weeks postinoculation.