Early Detection of Airborne Inoculum of Nothopassalora personata in Spore Trap Samples from Peanut Fields Using Quantitative PCR

Abstract

A quantitative PCR (qPCR)-assay was developed to detect airborne inoculum of Nothopassalora personata, causal agent of late leaf spot (LLS) on peanut, collected with a modified impaction spore trap. The qPCR assay was able to consistently detect as few as 10 spores with purified DNA and 25 spores based on crude DNA extraction from rods. In 2019, two spore traps were placed in two peanut fields with a history of LLS. Sampling units were replaced every 2 to 4 days and tested with the developed qPCR assay, while plots were monitored for symptom development. The system detected inoculum 35 to 56 days before visual symptoms developed in the field, with detection related to environmental parameters affecting pathogen life-cycle and disease development. This study develops the framework of the qPCR spore trap system and represents the initial steps towards validation of the performance of the system for use as a decision support tool to complement integrated management of LLS.

Field-based recombinase polymerase amplification and lab-based qPCR assays for detection of Helicoverpa armigera

“…The H. zea samples used here were live moths collected from pheromone traps after one or two nights. Three crude extraction methods were evaluated for use on whole moths of H. zea : i) the method of using ‘squish’ buffer (10 mM tris, 0.5 mM EDTA, 12.5 mM NaCl) extraction (Perera et al 2015), ii) the method from Munir et al (2020) using NaOH-based extraction buffer (100 mM NaOH, 2mM EDTA, 1% Triton X-100) and neutralization buffer (Tris-HCl pH 2), and iii) the commercial crude DNA extraction kit QuickExtract DNA Extraction Solution (Epicentre, Madison, WI). Ten H. zea moths were used to test each extraction method.…”

“…Using the modified method of Munir et al (2020), the microvials containing NaOH-based extraction buffer were placed in boiling water for 5 min. The microvials were then spun at 12,000 g for 5 min before transferring 100 µl of supernatant from each tube into a 1.5-ml microcentrifuge tube that contained 100 µl of neutralization buffer.…”

“…The crude DNA sample of H. armigera was extracted using the same NaOH-based method described above for individual moth samples. The crude DNA samples of H. zea used in these experiments were obtained by adding whole moths (100) and NaOH-based extraction buffer (100 ml total, 1 ml per specimen; Munir et al 2020) to a portable blender and running for 2 cycles of 40 s. The mixture was poured into a 20.3 cm × 30.5 cm polypropylene heavy duty autoclave bag (Weber Scientific, Hamilton Township, NJ), and the top was partially sealed with a stomacher blender bag clip (Seward Inc., Bohemia, NY) to allow minimal airflow. The bag was submerged in boiling water for 5 min.…”

A Method for Capture and Detection of Crop Airborne Disease Spores Based on Microfluidic Chips and Micro Raman Spectroscopy

“…However, this method requires professionals to crack the spores under strict experimental conditions. Only through tedious processing can fungal spores be detected [ 8 , 9 ]. The existing microscopic image method can realize rapid detection and recognition through morphology, but it cannot accurately identify spores with similar shape and size.…”

LAMP for in-field quantitative assessments of airborne grapevine downy mildew inoculum

“…It is, therefore, essential to develop efficient, reliable and easy‐to‐use tools for monitoring pathogens directly in the air. A number of methods are available for pathogen monitoring, including airborne spore trapping, which is an effective method for the early detection of outbreaks before symptoms appear on the plot (Munir et al, 2020; Rahman et al, 2017; Thiessen et al, 2016). Such monitoring can improve plant disease forecasting models (Van der Heyden et al, 2021).…”