Improved DNA extraction and quantitative real-time PCR for genotyping Erysiphe necator and detecting the DMI fungicide resistance marker A495T, using single ascocarps

Abstract

DNA extraction from minute fungal samples is challenging in all genetic studies. Identification of genetic groups and population biology mostly rely on the laborious production of single conidium isolates or on field samples, including infected plant materials. This paper reports a simple and cost-effective protocol for DNA extraction from individual chasmothecia of Erysiphe necator for subsequent applications. It is a less laborious alternative for genotyping purposes than production and analysis of single conidium isolates or analysis of infected plant material from the field. Using the protocols described here for 186 E. necator samples tested, genetic groups A and B were assigned. Based on CYP51 sequences, all the samples belonged to group B, while TUB2 sequences exhibited SNPs also diagnostic for group A. Additionally, a quantitative real-time PCR detection method of single nucleotide polymorphism in the CYP51 gene associated with DMI fungicide resistance was applied. The A495T marker, associated with DMI resistance, and here reported for the first time from Hungary, was detected by quantitative real-time PCR assays and direct sequencing of CYP51. The methods developed in this study can be applied as routine tests to monitor powdery mildew populations for fungicide resistance and other genetic characteristics.

Comprehensive analyses of the occurrence of a fungicide resistance marker and the genetic structure in Erysiphe necator populations

“…The fact that we did not detect linkage disequilibrium in our dataset means that sexual reproduction of E. necator takes place regularly. Our data indicate that group A is also reproducing sexually, similarly to other results 16 , 25 , 32 .…”

“…However, samplings from different populations showed that sexual reproduction could happen in group A 25 , 32 , and, mating-type assays revealed that group A produced chasmothecia and viable ascospores in laboratory conditions 25 , 32 . DNA markers of group A in field-collected chasmothecia were also detected, suggesting that sexual reproduction is also possible in natural conditions 16 .…”

“…Some studies revealed that group A spread mainly in spring, at the beginning of the season, while group B caused epidemics later in the season 18 , 21 , 33 . In Hungary, however, group B was detected throughout the vegetation period, and group A was also reported from samples collected later in the growing season, and it was mostly present together with group B 16 , 17 . Thus, these results did not support the hypothesis of temporal separation of groups A and B.…”

“…The fungus is introduced to Europe, where two distinct genetic groups, designated as A and B, were identified first based on DNA-fingerprint techniques 20 , 21 . Afterwards single nucleotide polymorphisms (SNPs) were assigned to those genotypes, in sequences of the β-tubulin ( TUB2 ) 16 , 17 , 22 , translation elongation factor 1-a ( EF1 -a) 18 , 23 , 24 and eburicol 14α-demethylase ( CYP51 ) genes 21 , and also in the sequences of the nrDNA internal transcribed spacer (ITS) 21 and intergenic spacer (IGS) regions 18 .…”

Fungi Parasitizing Powdery Mildew Fungi: Ampelomyces Strains as Biocontrol Agents against Powdery Mildews

“…The sporulation of many PM anamorphs is intense (Figure 1A), and the produced conidia (Figure 1B) spread rapidly [29,30]. While regularly applied fungicides are used to control PM, its frequent and inadequate use can lead to the emergence of fungicide resistance [7,31,32], as demonstrated in cucurbit PM fungi [5,6,[33][34][35][36]. In addition, plant leaves retain fungicides that are not completely decomposed by microorganisms, and the fungicides may also have negative side effects on plant physiology [37] as well as biodiversity [38].…”