Simultaneous detection and identification of pathogenic fungi in wheat using a DNA macroarray

The detection of economically important pathogens is a key element in sustainable wheat production and a prerequisite for crop protection. The objective of the project was to develop a DNA macroarray for fast and cost-effective detection of nine pathogenic fungi in wheat: Fusarium graminearum, Fusarium culmorum, Fusarium poae, Microdochium nivale var. majus, Microdochium nivale var. nivale, Puccinia recondita, Septoria tritici, Septoria nodorum and Pyrenophora tritici-repentis. Methodically, a macroarray is similar to a microarray but without the need for expensive equipment. PCR labelled samples of DNA are hybridized to pathogen-specific oligonucleotides (probes) anchored to a solid support. A positive reaction between an amplicon and a perfectly matched oligonucleotide generates a chemiluminescent signal which can be detected by a plate reader. The macroarray is sensitive enough to detect single nucleotide polymorphism (SNPs). Sample analysis is simple, fast, cost-effective, fully automated and suitable for high throughput screening. In this project, the nine wheat pathogens were detected within 6 hours simultaneously in a single sample using between one to four different species-specific probes for each pathogen. Species-specific detector oligonucleotides were designed based on the β-tubulin and/or succinate dehydrogenase region of fungal DNA. The detection limit of the DNA macroarray technique particularly depends on the pathogen-specific oligonucleotides deployed. The necessity for monitoring pathogenic fungi in wheat production and for prediction of crop yield has been recognized for a long time. The DNA macroarray responds very sensitively and has the potential to recognize pathogenic fungi earlier with reference to the cultivation period than a conventional PCR. This means that the DNA macroarray can detect genomic DNA from fungi in a lower potency than the conventional PCR. One benefit of the DNA macroarray for detection of fungal pathogens in wheat is its increased specificity and the other its application to a large number of microorganisms which can be detected in a single assay. This technology has been proven to be relatively cost-effective compared with real-time PCR or microarrays. This project was financially supported by the Commission of Technology and Innovation CTI in Berne, Switzerland

Paralog re-emergence: a novel, historically contingent mechanism in the evolution of antimicrobial resistance

Evolution of resistance to drugs and pesticides poses a serious threat to human health and agricultural production. CYP51 encodes the target site of azole fungicides, widely used clinically and in agriculture. Azole resistance can evolve due to point mutations or overexpression of CYP51, and previous studies have shown that fungicide-resistant alleles have arisen by de novo mutation. Paralogs CYP51A and CYP51B are found in filamentous ascomycetes, but CYP51A has been lost from multiple lineages. Here, we show that in the barley pathogen Rhynchosporium commune, re-emergence of CYP51A constitutes a novel mechanism for the evolution of resistance to azoles. Pyrosequencing analysis of historical barley leaf samples from a unique long-term experiment from 1892 to 2008 indicates that the majority of the R. commune population lacked CYP51A until 1985, after which the frequency of CYP51A rapidly increased. Functional analysis demonstrates that CYP51A retains the same substrate as CYP51B, but with different transcriptional regulation. Phylogenetic analyses show that the origin of CYP51A far predates azole use, and newly sequenced Rhynchosporium genomes show CYP51A persisting in the R. commune lineage rather than being regained by horizontal gene transfer; therefore, CYP51A re-emergence provides an example of adaptation to novel compounds by selection from standing genetic variation

Mutagenesis and Functional Studies with Succinate Dehydrogenase Inhibitors in the Wheat Pathogen Mycosphaerella graminicola

A range of novel carboxamide fungicides, inhibitors of the succinate dehydrogenase enzyme (SDH, EC 1.3.5.1) is currently being introduced to the crop protection market. The aim of this study was to explore the impact of structurally distinct carboxamides on target site resistance development and to assess possible impact on fitness

Assessment of QoI resistance in Plasmopara viticola oospores

QoI fungicides, inhibitors of mitochondrial respiration at the Qo site of cytochrome b in the mitochondrial bc1 enzyme complex, are commonly applied in vineyards against Plasmopara viticola (Berk. & MA Curtis) Berl. & De Toni. Numerous treatments per year with QoI fungicides can lead to the selection of resistant strains in the pathogen population owing to the very specific and efficient mode of action. In order to evaluate the resistance risk and its development, two different methods, biological and molecular, were applied to measure the sensitivity of oospores differentiated in vineyards, both treated and untreated with azoxystrobin, from 2000 to 2004. Assays using oospores have the advantage of analysing the sensitivity of bulked samples randomly collected in vineyards, describing accurately the status of resistance at the end of the grapevine growing season. Both methods correlated well in describing the resistance situation in vineyards. QoI resistance was not observed in one vineyard never treated with QoI fungicides. In the vineyard where azoxystrobin had been used in mixture with folpet, the selection of QoI-resistant strains was lower, compared with using solely QoI. In vineyards where QoI treatments have been stopped, a decrease in resistance was generally observed

Evolution of QoI resistance in Plasmopara viticola oospores

QoI resistance in P. viticola was first detected in France and Italy in 1999. Molecular and biological assays have been carried out since 2000 in order to provide reliable methods of detecting and quantifying resistance. Oospores were collected in vineyards located in northern and southern Italy. QoI resistance was evaluated by the germination rate of oospores on azoxystrobin amended medium and the frequency of mutant alleles in the DNA extracted from oospores. Both methods correlated to each other and were used side by side to test QoI resistance. Due to the spontaneous occurrence of the G143A mutation in wild type populations and the immigration from surrounding vineyards, resistance frequencies up to 10% were found in samples collected from vineyards never treated with QoIs. Particularly high values, about 90%, were associated with the application of five to six QoI treatments within the same season, while lower percentages, about 30%, were detected in vineyards treated with QoI used in mixture with fungicides belonging to a different resistance group. A progressive decrease of resistance frequency was observed when QoI applications were reduced in number or completely suspended for at least one season. Therefore, a full recovery of sensitivity may be achieved even in vineyards characterized by high levels of resistance, if particular care is taken during disease control by using QoIs only in mixtures and reducing the number of QoI treatments