Molecular Characterization and Detection of Mutations Associated with Resistance to Succinate Dehydrogenase-Inhibiting Fungicides in Alternaria solani

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 the primary means of achieving disease control. Previous work in our laboratory reported resistance to the succinate dehydrogenase-inhibiting (SDHI) fungicide boscalid in this plant pathogen with a concomitant loss of disease control. Two phenotypes were detected, one in which A. solani isolates were 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 exchanges 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 (50% effective concentration [EC50] < 5 μg ml(-1)), moderately resistant (EC50 = 5.1 to 20 μg ml(-1)), highly resistant (EC50 = 20.1 to 100 μg ml(-1)), and very highly resistant (EC50 > 100 μg ml(-1)) A. solani isolates. In total, five mutations were detected, two in each of the AsSdhB and AsSdhD genes and one in the AsSdhC gene. The sequencing of AsSdhB elucidated point mutations cytosine (C) to thymine (T) at nucleotide 990 and adenine (A) to guanine (G) at nucleotide 991, leading to an exchange from histidine to tyrosine (H278Y) or arginine (H278R), respectively, at codon 278. The H278R exchange was detected in 4 of 10 A. solani isolates moderately resistant to boscalid, exhibiting EC50 values of 6 to 8 μg ml(-1). Further genetic analysis also confirmed this mutation in isolates with high and very high EC50 values for boscalid of 28 to 500 μg ml(-1). Subsequent sequencing of AsSdhC and AsSdhD genes confirmed the presence of additional mutations from A to G at nucleotide position 490 in AsSdhC and at nucleotide position 398 in the AsSdhD, conferring H134R and H133R exchanges in AsSdhC and AsSdhD, respectively. The H134R exchange in AsSdhC was observed in A. solani isolates with sensitive, moderate, highly resistant, and very highly resistant boscalid phenotypes, and the AsSdhD H133R exchange was observed in isolates with both moderate and very high EC50 value boscalid phenotypes. Detection and differentiation of point mutations in AsSdhB resulting in H278R and H278Y exchanges in the AsSdhB subunit were facilitated by the development of a mismatch amplification mutation assay. Detection of these two mutations in boscalid-resistant isolates, in addition to mutations in AsSdhC and AsSdhD resulting in an H134R and H133R exchange, respectively, was achieved by the development of a multiplex polymerase chain reaction 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 exchanges but did contain a substitution of aspartate to glutamic acid at amino acid position 123 (D123E) in the AsSdhD subunit. Among A. solani isolates possessing resistance to boscalid, point mutations in AsSdhB were more frequently detected than mutations in genes coding for any other subunit.