Molecular Characterization and Diagnosis of QoI Resistance in Cucumber and Eggplant Fungal Pathogens

Abstract

The molecular mechanism of QoI fungicide resistance was studied using isolates of cucumber Corynespora leaf spot fungus (Corynespora cassiicola) and the eggplant leaf mold (Mycovellosiella nattrassii). In both pathogens, a mutation at position 143 from glycine to alanine (G143A) was detected in the cytochrome b gene that encodes for the fungicide-targeted protein. Moreover, the nucleotide sequence at amino acid position 143 was converted from GGT or GGA in sensitive (wild-type) to GCT or GCA in resistant (mutant-type) isolates. The methods of polymerase chain reaction restriction fragment length polymorphism commonly used for QoI resistance monitoring were employed successfully, leading to the amplified gene fragment from resistant isolates being cut with the restriction enzyme ItaI. However, heteroplasmy (the coexistence of wild-type and mutated alleles) was found when the resistant isolates of C. cassiicola, M. nattrassii, and Colletotrichum gloeosporioides (strawberry anthracnose fungus) were subcultured in the presence or absence of QoI fungicides. QoI resistance of cucumber powdery and downy mildew isolates persisted for a few years following the removal of the selection pressure imposed by the fungicide under both laboratory and commercial greenhouse conditions. The proportion of mutated sequences in cytochrome b gene decreased over time in the pathogen population. The protective efficacy of the full dose of azoxystrobin decreased when the populations of powdery and downy mildews contained resistant isolates at 10%. Using FMBIO, a fluorescence bio-imaging analyzer, the mutant allele from the QoI-resistant isolates could be detected at the level of 1%, whereas the detection sensitivity of ethidium-bromide-stained gels was approximately 10 times lower.

Fungicide-induced transposon movement in Monilinia fructicola

“…Such changes could include typical mechanisms that lead to fungicide resistance, such as the movement of transposable elements into the promoter region of a target gene (Luo and Schnabel, 2008b;Ma et al, 2006) or the implementation of nucleotide sequence variations in target genes (Ishii et al, 2007;Leroux et al, 2007). The purpose of this study was to investigate whether fungicide stress can lead to genetic changes typically associated with fungicide resistance in a phytopathogenic, filamentous fungus.…”

Detection of Fungicide Resistance

“…A more recent study by Lesniak et al (2011), found ninety-eight percent of QoIresistant/shifted isolates screened in Michigan were associated with G143A, consistent with other phytopathogenic fungi resistant to this class of fungicides (Ishii et al, 2009;Ishii et al 2007). Sequencing the entire CYTb gene of resistant isolates that did not test positive for the G143A transition did not reveal other mutations in the structural gene, and suggests other mechanisms of resistance (Lesniak et al 2011).…”

Mechanism of action and selectivity of a novel fungicide, pyribencarb

“…1) binds with a hydrogen bond to the amide group of glutamic acid at position 272 in domain C; therefore, this region plays an important role in accepting fungicides. Since substitution of glycine for alanine at position 143 (G143A) in domain B significantly lowers the affinity of strobilurin fungicides to cytochrome b, 7,8,13,14,[19][20][21][22][23] interaction between domain B and strobilurin fungicides must be strong. On the other hand, substitution of phenylalanine for leucine at position 129 (F129L) in domain A has been shown not to significantly lower the affinity of strobilurin fungicides to cytochrome b.…”

“…These strains have been elucidated to have a substitution of glycine for alanine at position 143 (G143A mutation) in cytochrome b. 5,8) Fischer rats F344/DuCrlCrlj (Rattus norvegicus) and carp (Cyprinus carpio) were obtained from Charles River Laboratories Japan Inc. (Kanagawa, Japan) and Kato Koi Farm Company (Gifu, Japan), respectively. Etiolated seedlings of crops [cucumber (Cucumis sativus L.), tomato (Lycopersicon esculentum Mill.…”