Ontogenic scab resistance in apple leaves and fruits is a horizontal resistance against the plant pathogen Venturia inaequalis and is expressed as a decrease in disease symptoms and incidence with the ageing of the leaves. Several studies at the biochemical level tried to unveil the nature of this resistance; however, no conclusive results were reported. We decided therefore to investigate the genetic origin of this phenomenon by performing a full quantitative transcriptome sequencing and comparison of young (susceptible) and old (ontogenic resistant) leaves, infected or not with the pathogen. Two time points at 72 and 96 hours post-inoculation were chosen for RNA sampling and sequencing. Comparison between the different conditions (young and old leaves, inoculated or not) should allow the identification of differentially expressed genes which may represent different induced plant defence reactions leading to ontogenic resistance or may be the cause of a constitutive (uninoculated with the pathogen) shift toward resistance in old leaves. Differentially expressed genes were then characterised for their function by homology to A. thaliana and other plant genes, particularly looking for genes involved in pathways already suspected of appertaining to ontogenic resistance in apple or other hosts, or to plant defence mechanisms in general.In this work, five candidate genes putatively involved in the ontogenic resistance of apple were identified: a gene encoding an “enhanced disease susceptibility 1 protein” was found to be down-regulated in both uninoculated and inoculated old leaves at 96 hpi, while the other four genes encoding proteins (metallothionein3-like protein, lipoxygenase, lipid transfer protein, and a peroxidase 3) were found to be constitutively up-regulated in inoculated and uninoculated old leaves. The modulation of the five candidate genes has been validated using the real-time quantitative PCR. Thus, ontogenic resistance may be the result of the corresponding up- and down-regulation of these genes.

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Fire blight (FB), caused by Erwinia amylovora, is one of the most important pome fruit pathogens worldwide. To control this devastating disease, various chemical and biological treatments are commonly applied in Switzerland, but they fail to keep the infection at an acceptable level in years of heavy disease pressure. The Swiss authorities therefore currently allow the controlled use of the antibiotic streptomycin against FB in years that are predicted to have heavy infection periods, but only one treatment per season is permitted. Another strategy for controlling Erwinia is to breed resistant/tolerant apple cultivars. One way of accelerating the breeding process is to obtain resistant cultivars by inserting one or several major resistance genes, using genetic engineering. To date, no study summarizing the impact of different FB control measures on the environment and on human health has been performed. This study consequently aims to compare different disease-control measures (biological control, chemical control, control by antibiotics and by resistant/tolerant apple cultivars obtained through conventional or molecular breeding) applied against E. amylovora, considering different protection goals (protection of human health, environment, agricultural diversity and economic interest), with special emphasis on biosafety aspects. Information on each FB control measure in relation to the specified protection goal was assessed by literature searches and by interviews with experts. Based on our results it can be concluded that the FB control measures currently applied in Switzerland are safe for consumers, workers and the environment. However, there are several gaps in our knowledge of the human health and environmental impacts analyzed: data are missing (1) on long term studies on the efficacy of most of the analyzed FB control measures; (2) on the safety of operators handling streptomycin; (3) on residue analyses of Equisetum plant extract, the copper and aluminum compounds used in apple production; and (4) on the effect of biological and chemical control measures on non-target fauna and flora. These gaps urgently need to be addressed in the near future.

Gusberti, M., Patocchi, A., Gessler, C, and Broggini, G. A. L. 2012. Quantification of Venturia inaequalis growth in Malus x domestica with quantitative real-time polymerase chain reaction. Plant Dis. 96:1791-1797.A quantitative real-time polymerase chain reaction (qPCR) was developed and validated for quantification of Venturia inaequalis in infected leaf tissue of Malus x domestica. The method is based on dual-labeled hybridization probes, allowing simuhaneous detection of host and pathogen DNA within one single reaction. Limit of quantification for the pathogen was 0.5 pg per reaction and, for the host, reached 5 pg per reaction. The fungal growth measured in four apple cultivars 2 weeks after inoculation significantly correlated with their different level of scab resistance and allowed the observation of ontogenic resistance. After sporulation on the youngest leaf, fungal biomass in susceptible 'Gala' was 118 dmes higher than in resistant 'Fiorina' and 'Discovery' while intermediate values were found with the intermediate susceptible 'Milwa'. Correlation was also observed between severity classes obtained by visual scoring of symptoms and qPCR results. Moreover, qPCR demonstrated validity of the developed method as a disease severity forecast tool 10 days after the pathogen's inoculation and prior to the appearance of the symptoms. Applications of the methodology can include the quantification of scab resistance during breeding programs, evaluation of fungicide and biocontrol efficacy, and quantification of the fitness of different pathogenic strains.

The expression of the apple scab resistance gene Rvi6 in different apple cultivars and lines is not modulated by biotic or abiotic factors. All commercially important apple cultivars are susceptible to Venturia inaequalis, the causal organism of apple scab. A limited number of apple cultivars were bred to express the resistance gene Vf from the wild apple genotype Malus floribunda 821. Positional cloning of the Vf locus allowed the identification of the Rvi6 (formerly HcrVf2) scab resistance gene that was subsequently used to generate cisgenic apple lines. It is important to understand and compare how this resistance gene is transcribed and modulated during infection in conventionally bred cultivars and in cisgenic lines. The aim of this work was to study the transcription pattern of Rvi6 in three classically bred apple cultivars and six lines of 'Gala' genetically modified to express Rvi6. Rvi6 transcription was analyzed at two time points using quantitative real-time PCR (RT-qPCR) following inoculation with V. inaequalis conidia or water. Rvi6 transcription was assessed in relation to five reference genes. β-Actin, RNAPol, and UBC were the most suited to performing RT-qPCR experiments on Malus × domestica. Inoculation with V. inaequalis conidia under conditions conducive to scab infection failed to produce any significant changes to the transcription level of Rvi6. Rvi6 expression levels were inconsistent in response to external treatments in the different apple cultivars, and transgenic, intragenic or cisgenic lines.