Identification of the mutation responsible for resistance to QoI fungicides and its detection in Ascochyta rabiei (teleomorph Didymella rabiei)

Abstract

This study characterized a fragment of the cytochrome b gene from Ascochyta rabiei isolates collected in North Dakota, USA, that varied in sensitivity to quinone-outside inhibitor (QoI) fungicides. The sequenced genomic DNA fragment contained a group I intron immediately after codon 131. The size of the cytochrome b gene was estimated to be over 4AE6 kb. Multiple alignment analysis of cDNA and protein sequences revealed a mutation that changed the codon for amino acid 143 from GGT to GCT, introducing an amino acid substitution from glycine to alanine (G143A), which is frequently associated with QoI resistance. Based on this mutation, a diagnostic PCR assay was developed using an approach called mismatch amplification mutation assay. This method was successfully validated by testing a total of 70 A. rabiei isolates, of which 38 isolates were found to be QoI-resistant. This fast and accurate PCR assay provides a very useful and simple screening method for QoI resistance in A. rabiei isolates.

The Detection and Characterization of QoI-Resistant Didymella rabiei Causing Ascochyta Blight of Chickpea in Montana

“…Other researchers have been successful in developing a molecular technique to detect the G143A mutation (Delgado et al, 2013). This is a qualitative assay that cannot be used to monitor the frequency of G143 and A143 alleles both in individual isolates and in mixed field populations.…”

“…Currently, of the three classes of fungicide, QoI fungicides are the choice of most pulse growers for pre- and post-infection management of AB in the United States and Canada ( Wise et al, 2008 ; Delgado et al, 2013 ; Lonergan et al, 2015 ; Bowness et al, 2016 ). Prior to 2007, it was the only available fungicide MOA on pulse crops and resistance developed rapidly in North Dakota and Canada ( Gossen and Anderson, 2004 ; Wise et al, 2011 ; Bowness et al, 2016 ).…”

“…The fungicide binds to the center of the quinone (Qo) site of the cytochrome bc1 complex (complex III) on the positive side of the inner mitochondrial membrane. This causes depletion of adenosine triphosphate (ATP) that ultimately halts spore germination due to energy inadequacy ( Grasso et al, 2006a ; Wise et al, 2009 ; Delgado et al, 2013 ).…”

“…tritici, Microdochium majus, Ourcosphaerella graminicola , ( Sierotzki et al, 2000 ; Amand et al, 2003 ; Walker et al, 2009 ; Patel et al, 2012 ), and several other fungal pathogens including Cercospora sojinia, Colletotrichum graminicola, Alternaria alternata, Botrytis cinerea, Pyricularia grisea, Podosphaera fusca, Pythium aphanidermatum, Pyrenophora teres , and Pseudoperonospora cubensis ( Ishii et al, 2001 ; Gisi et al, 2002 ; Avila-Adame et al, 2003 ; Kim et al, 2003 ; Ma et al, 2003 ; Sierotzki et al, 2007 ; Banno et al, 2009 ; Samuel et al, 2011 ; Zeng et al, 2015 ;). In addition, QoI resistance has been reported in D. rabiei in North Dakota and Canada ( Gossen and Anderson, 2004 ; Wise et al, 2009 ; Delgado et al, 2013 ). “The mechanism of resistance of D. rabiei has been attributed to single amino acid replacement in the cytochrome b protein of the cytochrome bc1 complex” ( Delgado et al, 2013 ).…”

“…In addition, QoI resistance has been reported in D. rabiei in North Dakota and Canada ( Gossen and Anderson, 2004 ; Wise et al, 2009 ; Delgado et al, 2013 ). “The mechanism of resistance of D. rabiei has been attributed to single amino acid replacement in the cytochrome b protein of the cytochrome bc1 complex” ( Delgado et al, 2013 ). Currently, three amino acid substitutions are found in the cytochrome b protein of fungal plant pathogens that confer different degrees of resistance to QoI fungicides ( Grasso et al, 2006a ; Delgado et al, 2013 ).…”