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Detection of Podosphaera macularis in Air Samples by Quantitative PCR
David H Gent1,2, Nanci L Adair2, Ross J Hatlen3
1Department of Botany and Plant Pathology, Oregon State University, Corvallis, OR 97331.
Abstract:
Detection and quantification of pathogen propagules in the air or other environmental samples is facilitated by culture-independent assays. We developed a quantitative PCR assay for the hop powdery mildew fungus, Podosphaera macularis, for detection of the organism from air samples. The assay uses primers and a TaqMan probe designed to target species-specific sequences in the 28S large subunit of the nuclear ribosomal DNA. Analytical sensitivity was not affected by the presence of an exogenous internal control or potential PCR inhibitors associated with DNA extracted from soil. The level of quantification of the assay was between 200 and 350 conidia when DNA was extracted from a fixed number of conidia. The assay amplified all isolates of P. macularis tested and had minimal cross-reactivity with other Podosphaera species when assayed with biologically relevant quantities of DNA. Standard curves generated independently in two other laboratories indicated that assay sensitivity was qualitatively similar and reproducible. All laboratories successfully detected eight unknown isolates of P. macularis and correctly discriminated Pseudoperonospora humuli and a water control. The usefulness of the assay for air sampling for late-season inoculum of P. macularis was demonstrated in field studies in 2019 and 2020. In both years, airborne populations of P. macularis in hop yards were detected consistently and increased during bloom and cone development.
Insights
A new quantitative PCR assay accurately detects and quantifies hop powdery mildew fungus (Podosphaera macularis) in air samples. This method is reproducible and effective for monitoring airborne pathogen populations in hop yards.
Area of Science:
- Plant Pathology
- Molecular Biology
- Agricultural Science
Background:
- Culture-independent assays are crucial for detecting airborne pathogens.
- Hop powdery mildew, caused by Podosphaera macularis, significantly impacts crop yield.
- Accurate quantification of airborne inoculum is needed for effective disease management.
Purpose of the Study:
- To develop and validate a quantitative PCR (qPCR) assay for detecting Podosphaera macularis in air samples.
- To assess the assay's sensitivity, specificity, and reproducibility.
- To evaluate the assay's utility in field conditions for monitoring airborne inoculum.
Main Methods:
- Developed a qPCR assay targeting species-specific sequences in the 28S nuclear ribosomal DNA of Podosphaera macularis.
- Tested analytical sensitivity with varying conidial loads and in the presence of inhibitors.
- Assessed specificity against related Podosphaera species and field isolates.
- Validated assay reproducibility across three independent laboratories using standard curves and unknown samples.
- Conducted field studies in hop yards to monitor airborne P. macularis populations.
Main Results:
- The qPCR assay demonstrated high analytical sensitivity, quantifying down to 200-350 conidia.
- The assay showed minimal cross-reactivity with other Podosphaera species and accurately detected P. macularis isolates.
- Reproducibility was confirmed across laboratories, with successful detection and discrimination of target and non-target organisms.
- Field studies consistently detected airborne P. macularis, with populations increasing during hop bloom and cone development.
Conclusions:
- The developed qPCR assay is a sensitive, specific, and reproducible tool for detecting and quantifying airborne Podosphaera macularis.
- This assay is suitable for environmental monitoring, particularly for air sampling in hop yards.
- The findings support the use of this qPCR assay for early detection and management of hop powdery mildew.
