Detecting the asymptomatic colonization of apple branches by Neonectria ditissima, causing European canker of apple

Abstract

European canker is one of the most devastating fungal diseases of apple in most temperate regions. The causal agent, Neonectria ditissima, infects trees through wounds in the bark forming cankers that girdle the stem and eventually cause tree death. Timely protection of the trees is challenged by stagnation of symptom expression after infections for a long period of time. The objective of this research is to use a novel TaqMan PCR assay to detect and quantify N. ditissima during the asymptomatic colonization of apple wood. Pruning wounds on branches of the cultivars Elstar and Gala were inoculated with N. ditissima and wood discs were sampled at 2–6, 10–14, and 30–34 mm distance from the inoculation site after 3 hours, 2 weeks, 4 weeks and 8 weeks for the detection and quantification of the pathogen. The TaqMan PCR assay detected N. ditissima in 51% of the inoculated apple tree samples. This was more sensitive than the culturing method detecting N. ditissima in 11% of the samples. An accumulation of N. ditissima DNA up to 34 mm distance from the inoculation site was observed without development of visible symptoms. To our knowledge this is the first time colonization of N. ditissima was detected and quantified in the absence of symptoms of European canker. The implications of this research are discussed.

From amplicons to strains: The limitations of metabarcoding as criterium in the selection process of biocontrol strains against the pome fruit pathogenNeonectria ditissima

“…The experiment using potted tress and the sample collection were described in detail in Harteveld et al [23]. In short: 40 2-year-old potted trees of cultivars ‘Elstar’ (moderately resistant) and ‘Gala’ (susceptible) were used to study progression of N. ditissima .…”

“…To make more accurate correlations, microbial compositions had to be linked to absolute N. ditissima abundances. Due to the TaqMan results published in Harteveld et al (2023) [23], absolute abundances of N. ditissima were available for each sample. In addition, relative abundance of the pathogen per sample could be derived from the microbiome analysis.…”

“…However, there was no direct link between the microbiome and the pathogen in these studies. To this end we combined a community analysis of bacterial 16S and fungal ITS barcodes with a study lead by Harteveld et al (2023) on colonization of apple branches by N. ditissima and the results on antagonistic fungi against N. ditissima isolated by Elena et al (2022) from infected apple branches [18,23].…”

“…The aim of this current study is to combine the knowledge on the microbiome of the host plant in selected samples taken during the study of Harteveld et al [23], using 16S and ITS metabarcoding with quantitative data on the N. ditissima branch colonisation to see if microbial components that show negative or positive correlations with N. ditissima concentrations can be identified. In a next step, ITS amplicons from the metabarcoding and those of the collection of potential antagonists against N. ditissima acquired in the study of Elena et al (2022) were compared to evaluate if and how metabarcoding analysis can support decisions on selection of biological control agents [18].…”