Tan spot, which is a significant foliar disease of wheat causing severe yield loss in many wheat-growing areas worldwide, is caused by the fungus Pyrenophora tritici-repentis (PTR). The pathogenicity of PTR mainly depends upon the production of host selective toxins (HSTs). Consequently, phenotypic race identification and production of HSTs were assessed using 42 PTR isolates on five wheat differentials. PTR race identification revealed the presence of Race 1 (86%) and Race 3 (14%) in Oklahoma. Ptr ToxA is a major necrosis-inducing HST found in almost all PTR isolates in Oklahoma. Therefore, two PTR isolates, possessing (+) and lacking (-) the ToxA gene, were transformed to express fluorescent proteins to examine the infection and colonization process on susceptible and resistant wheat differentials. Results showed that fungal penetration and growth for ToxA+ and toxa- isolates started at 3DPI. On susceptible cultivars, ToxA+ isolates produced necrosis at 5DPI and 7DPI, consistent with the formation of secondary hyphae growing directly into epidermal and mesophyll cells, however on resistant cultivars, intracellular hyphae remain restricted, producing reduced infection sites with intense auto-fluorescent borders, which was also seen in susceptible and resistant wheat cultivars, when inoculated with toxa- isolates. Additionally, spray-induced gene silencing (SIGS) and virus-induced gene silencing (VIGS) were evaluated to reduce disease development by silencing the ToxA gene. Co-incubation of the fungal spores with Cy3-labeled dsRNA control demonstrated that PTR could take up exogenous dsRNA. However, SIGS appears unsuitable against PTR since all our attempts to silence multiple genes resulted in a lack of gene silencing. However, gene silencing was achieved using BMV: VIGS vectors targeting five regions of the ToxA gene. RT-qPCR revealed a significant reduction in the ToxA transcripts after the infection with the pC13/F3CP5 vector with all the ToxA inserts, compared to the control, infiltration buffer, and empty vector. Overall, more than 50% reduction of the target mRNA was detected in all the BMV: VIGS vectors. Additionally, a negative correlation between fragment size and silencing efficiency was determined. These results will provide insight for developing and improving management methods to reduce disease infection of PTR.
Salome E. Suarez Freile (Sat,) studied this question.