Multiple mutations across the succinate dehydrogenase gene complex are associated with boscalid resistance in Didymella tanaceti in pyrethrum

Authors: PEARCE, TAMIEKA - University Of Tasmania; SCOTT, JASON - University Of Tasmania; WILSON, CALUM - University Of Tasmania; Gent, David - Dave; PETHYBRIDGE, SARAH - Cornell University; HAY, FRANK - Cornell University

Submitted to: PLOS ONE
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 6/5/2019
Publication Date: 6/20/2019
Citation: Pearce, T.L., Scott, J.B., Wilson, C.R., Gent, D.H., Pethybridge, S.J., Hay, F.S. 2019. Multiple mutations across the succinate dehydrogenase gene complex are associated with boscalid resistance in Didymella tanaceti in pyrethrum. PLoS One. 14(6):e0218569. https://doi.org/10.1371/journal.pone.0218569.
DOI: https://doi.org/10.1371/journal.pone.0218569

Interpretive Summary: Tan spot is an important disease of pyrethrum, which is the source of naturally occurring insecticidal compounds known as pyrethrins. Disease control failures with tan spot have been associated with decreased sensitivity of the fungus to the SDHI fungicide boscalid. In this research, genetic mutations were identified in the Sdh gene associated with varying levels of sensitivity to the fungicide. A total of 16 different mutations were found, which resulted in varying levels of fungicide resistance. Further, a rapid assay for detecting the mutations was developed. In total, this research has discovered that the genetic basis of resistance to the fungicide boscalid is complex in the tan spot fungus. Strategies for mitigation of fungicide resistance are proposed.

Technical Abstract: Decreased control of tan spot of pyrethrum, caused by Didymella tanaceti, has been attributed to decreased sensitivity of the pathogen population to the SDHI fungicide boscalid. Sequencing the SdhB, SdhC, and SdhD of isolates with resistant and sensitive phenotypes identified 16 mutations, resulting in three substitutions in the Sdh B (H277Y/R, I279V), six substitutions in the SdhC (S73P, G79R, H134R, H134Q, S135R and combined H134Q/S135R), and two substitutions in the SdhD (D112E, H122R). In vitro boscalid testing was used to calculate EC50 and resistance factors (RF) for isolates with each substitution . All isolates with wild-type (WT) SDH profiles were sensitive to boscalid (RF < 2.2) while isolates with substitutions had RF indicating moderate to very high resistance phenotypes. Isolates with SdhB-I279V, SdhC-H134Q and SdhD-D112E exhibited a moderate resistance phenotype (10 =RF = 100) and isolates with SdhC-H134R were associated with a very high resistance phenotype (RF = 1000). All other substitutions exhibited a high resistance phenotype (100 = RF = 1000). A high-resolution melt assay was designed to analyse the frequency of Sdh mutations in four field populations (n = 774) collected in 2012. Prior to boscalid application in August, 92.9% of D. tanaceti contained a substitution associated with decreased sensitivity in the SdhB, SdhC or SdhD. Following boscalid application, 98.9% contained a substitution associated with decreased sensitivity and a significant increase in the frequency of SdhB-H277Y/R and decrease in WT isolates. WT isolates were not detected in three of four fields evaluated following fungicide application. The SdhB-H277Y, SdhC-H134R and SdhB-H277R genotypes were most frequently observed in the population at 56.7, 19.0, and 10.3%, respectively. Previously obtained microsatellite and mating-type data was used to evaluate the genetic diversity within isolates possessing each SDH substitution. No substitution consisted entirely of clones.