Monitoring of Venturia inaequalis harbouring the QoI resistance G143A mutation in French orchards as revealed by PCR assays

Detection of Fungicide Resistance

“…Numerous books and laboratory manuals exist as references for PCR operation and optimization (Sambrook et al, 1989). PCR based detection of fungicide resistance depends upon the ability of the reaction to selectively amplify specific regions of DNA, and usually require several post-PCR steps, including agarose gel electrophoresis for either confirmation of amplicon presence or size, or restriction enzyme analysis (Lesniak et al 2011;Fontaine et al 2009;Quello et al 2009; and reviewed by Ma and Michailides 2005). The development of new fluorescent techniques (LAMP, etc) has led to novel assay formats that greatly simplify the protocols used for the detection of specific nucleic acid sequences (Nurmi et al 2000) and allow for the detection of a specific PCR product in a homogeneous solution without the need to open the amplification tubes after PCR or gel electrophoresis (Tomlinson et al 2012).…”

“…For most, smaller laboratories, a more realistic approach concerns the use of polymerase chain reaction coupled with cleaved amplified polymorphic sequences (PCR-CAPS). Restriction-fragment length polymorphisms due to small nucleotide polymorphisms (SNP) that co-segregate or are caused by fungicide resistance create or abolish restriction sites in PCR products, and can be exploited for detection of fungicide resistance through the careful selection of locus-specific oligonucleotide primers (Banno et al 2008;Lesemann et al 2007, Quello et al 2009, Fontaine et al 2009others).…”

“…An alternative to PCR-CAPS is allele specific (AS-PCR), in which the mutation that confers resistance is used to design primers that specifically amplify the mutated allele, but not the wild-type one (Fontaine et al 2009, Lesniak et al 2011. This approach is more sensitive, and does not require a RFLP to detect resistance, but also does not detect any heteroplasmy or heterozygosity.…”

Disease Decision Support Systems: Their Impact on Disease Management and Durability of Fungicide Effectiveness

“…Resistance to multisite inhibitors, DMIs, anilinopyrimidines and SDHIs are still monitored with biological tests. For benzimidazoles and QoIs, resistance was found to be determined by single mutation within the target genes (β-tubulin and cytochrome b, respectively) (Quello et al, 2010;Fontaine et al, 2008). These mutations were detected after partial PCR amplification of these genes and digestion of the PCR product by a restriction enzyme that recognizes the restriction site generated (or lost) by the mutation determining resistance (PCR-RFLP or CAPS test).…”

“…This test was developed for mono-lesion isolates, in the case of benzimidazoles and for bulk population of spores in the case of QoIs. Moreover, an allelespecific PCR test (AS-PCR) was also developed to detect the G143A change within the cytochrome b of V. inaequalis (Fontaine et al, 2008). Roughly, PCR was achieved using primers differing by their 3' nucleotides, enabling to detect specifically either the susceptible or the resistant allele of the sample.…”