A method to monitor airborne Venturia inaequalis ascospores using volumetric spore traps and quantitative PCR

Abstract

Apple scab caused by the fungus Venturia inaequalis can result in significant crop losses if not managed effectively. Sanitation as part of an integrated management strategy aims to significantly reduce primary inoculum to lower the disease pressure. This study evaluates the possibility of molecular detection and quantification of ascospore discharge and the use of this method to test for efficacy of orchard sanitation treatments. A method to detect and quantify airborne ascospores was developed using volumetric spore traps (VSTs). V. inaequalis specific primers were tested on daily VST samples from two orchard sections (leaf litter removed compared to leaf litter left) during spring. A molecular method to detect and quantify ascospores was tested by amplifying genomic regions of the mitochondrial CYP51A1 gene, and the ITS region using SYBR® green. Timing of ascospore discharge was compared to predicted infection risk and a degree day model using weather data. The average spore detection limit was estimated to be at levels of 1 pg μl −1 DNA (approximately 37 ascospores) per daily spore trap reading using CYP51A1 primers. Using the CYP51A1 primer pair, primary inoculum was estimated to be 51 % lower in the orchard sections where leaves had been removed, indicating that this method could be used to evaluate the efficacy of alternative control strategies such as leaf removal to reduce potential ascospore dose. This is the first report of combining VSTs and quantitative PCR to monitor airborne V. inaequalis ascospores.

New Molecular Tool for a Quick and Easy Detection of Apple Scab in the Field

“…Another key tool for monitoring V. inaequalis is spore trap devices [26][27][28][29]. Ascospores are captured on tapes in the field and then identified and quantified using microscope observation [25].…”

“…Molecular methods overcome microscope observation in terms of sensitivity and specificity, as well as for speed of the analysis, and they have been widely used to monitor the presence of ascospores [28][29][30]. PCR-based methods for some species of Venturia have been designed on the sequence of internal transcribed spacer (ITS) or on the cytochrome 51A1 (CYP51A1) [31,32], while qPCR assays have been designed on a broader range of molecular regions including the ITS rDNA, ATP-binding cassette transporter 2 (ABC2), CYP51A1 and EF-1α genes [28][29][30]33]. qPCR techniques have been used in combination with spore trap sampling, although their application requires a centralised laboratory, and the interpretation of the results is laborious.…”

Molecular methods for the detection and quantification of Pestalotiopsis and Neopestalotiopsis inoculum associated with macadamia

“…Variation in inoculum load over time in orchards could be rapidly assessed by this method. qPCR systems coupled with spore trapping have been used to estimate the airborne inoculum of other fungal pathogens, such as Phyllosticta citricarpa in citrus (Tran et al, 2020), downy mildew pathogens of spinach and beet (Klosterman et al, 2014), Venturia inaequalis in apple (Meitz-Hopkins et al, 2014), Mycosphaerella graminicola in wheat (Duvivier et al, 2013), and Botrytis squamosa in onion (Carisse et al, 2009). The newly developed qPCR system could be an alternative method to conventional methods relying on microscopy.…”

Influence of climatic factors on dry flower, grey and green mould diseases of macadamia flowers in Australia

“…Real-time quantitative PCR (qPCR) is an effective tool used to detect and quantify airborne conidia. Spore trapping combined with qPCR method has been used for the detection and quantification of several tree fungal pathogens, such as Venturia inaequalis (Meitz-Hopkins et al, 2014), Hymenoscyphus pseudoalbidus (Chandelier et al, 2014) and Fusarium circinatum (Quesada et al, 2018). Given the vital role wind-borne conidia play in disease spread, knowledge of temporal dispersal of the flower blight fungal inoculum during the flowering would provide useful information that may aid disease management decisions.…”