Page Banner

United States Department of Agriculture

Agricultural Research Service

Research Project: Molecular Approaches for Improved Cercospora Leaf Spot Control in Sugarbeet

Location: Sugarbeet and Potato Research

Title: Molecular characterization and detection of mutations associated with resistance to succinate dehydrogenase inhibiting (SDHI) fungicides in Alternaria solani

Authors
item Mallik, Ipsita -
item Arabiat, Sahar -
item Pasche, Julie -
item Bolton, Melvin
item Patel, Jaimin -
item Gudmestad, Neil -

Submitted to: Phytopathology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: July 19, 2013
Publication Date: January 1, 2014
Citation: Mallik, I., Arabiat, S., Pasche, J.S., Bolton, M.D., Patel, J.S., Gudmestad, N.C. 2014. Molecular characterization and detection of mutations associated with resistance to succinate dehydrogenase inhibiting (SDHI) fungicides in Alternaria solani. Phytopathology. 104(1):40-49.

Interpretive Summary: Early blight, caused by Alternaria solani, is an economically important foliar disease of potato in several production areas of the United States. Few potato cultivars possess resistance to early blight, therefore, the application of fungicides is a primary means of achieving disease control. Previous work in our laboratory reported resistance to the succinate dehydrogenase inhibiting (SDHII) fungicide boscalid in this plant pathogen. Two types of resistant isolates were detected, one in which A. solani isolates appear to be moderately resistant to boscalid, the other in which isolates were highly resistant to the fungicide. Resistance in other fungal plant pathogens to SDHI fungicides is known to occur due to specific changes in proteins named succinate dehydrogenase B (SdhB), C (SdhC), and D (SdhD). In this study, the genes that encode these proteins were analyzed and compared in fungicide sensitive, moderately resistant, highly resistant, and very highly resistant A. solani isolates. The sequencing of AsSdhB, which encodes the SdhB protein, identified mutations in this gene called H278R and H278Y. The H278R mutation was associated most commonly with A. solani isolates moderately resistant to boscalid, although further genetic analysis also confirmed this mutation in isolates with high and very high EC50 values to boscalid. The H278Y mutation was commonly found in isolates highly and very highly resistant to boscalid. Further sequencing confirmed the presence of additional mutations called H134R and H133R in the genes that encode SdhC and SdhD, respectively. The H134R mutation was most commonly observed in moderately resistant A. solani isolates while the H133R mutation was observed in isolates that were both moderate and very highly resistant to boscalid. Detection and differentiation of the H278R and H278Y mutations were facilitated by the development of a mismatch amplification mutation assay. Detection of these two mutations, in addition to H134R and H133R was achieved by the development of a multiplex PCR to detect and differentiate the sensitive and resistant isolates based on the single mutation present in all three genes. A single A. solani isolate with resistance to boscalid did not contain any of the above mentioned mutations, but did contain a mutation called D123E in the SdhD protein. Among A. solani isolates possessing resistance to boscalid, mutations in SdhB were more frequently detected than any other mutation.

Technical Abstract: Early blight, caused by Alternaria solani, is an economically important foliar disease of potato in several production areas of the United States. Few potato cultivars possess resistance to early blight, therefore, the application of fungicides is a primary means of achieving disease control. Previous work in our laboratory reported resistance to the succinate dehydrogenase inhibiting (SDHII) fungicide boscalid in this plant pathogen. Two phenotypes were detected, one in which A. solani isolates appear to be moderately resistant to boscalid, the other in which isolates were highly resistant to the fungicide. Resistance in other fungal plant pathogens to SDHI fungicides is known to occur due to amino acid transitions in the soluble subunit succinate dehydrogenase B (SdhB), C (SdhC), and D (SdhD) proteins. In this study, the AsSdhB, AsSdhC, and AsSdhD genes were analyzed and compared in sensitive (EC50 < 2 'g ml-1), moderately resistant (EC50 = 2-10 'g ml-1), highly resistant (EC50 = 10.1-100 'g ml-1) and very highly resistant (EC50 > 100 'g ml-1) A. solani isolates. The sequencing of AsSdhB elucidated point mutations leading to a transition at codon 278 from either histidine to arginine (H278R) or histidine to tyrosine (H278Y). The H278R mutation was associated most commonly with A. solani isolates moderately resistant to boscalid, exhibiting EC50 values ranging from 3 'g ml-1 to 8 'g ml-1, although further genetic analysis also confirmed this mutation in isolates with high and very high EC50 values to boscalid. The H278Y mutation was commonly found in isolates highly and very highly resistant to boscalid with EC50 values ranging from 13 'g ml-1 to 500 'g ml-1. Further sequencing confirmed the presence of additional mutations conferring H134R and H133R transitions in AsSdhC and AsSdhD, respectively. The H134R mutation in AsSdhC was most commonly observed in A. solani isolates with moderately resistant boscalid EC50 values and the AsSdhD-H133R mutation was observed in isolates with both moderate and very high EC50 values. Detection and differentiation of the H278R and H278Y point mutations in the AsSdhB subunit were facilitated by the development of a mismatch amplification mutation assay. Detection of these two mutations, in addition to the H134R mutation in the AsSdhC subunit and H133R mutation in the AsSdhD subunit, was achieved by the development of a multiplex PCR to detect and differentiate the sensitive and resistant isolates based on the single nucleotide polymorphisms present in all three genes. A single A. solani isolate with resistance to boscalid did not contain any of the above mentioned mutations, but did contain a substitution of aspartate to glutamic acid at amino acid position 123 (D123E) in AsSdhD subunit. Among A. solani isolates possessing resistance to boscalid, point mutations in AsSdhB were more frequently detected than any other mutation.

Last Modified: 8/27/2014
Footer Content Back to Top of Page