Volume 19 Part 1 Article 19: Sampling and detection of Dry Bubble (Lecanicillium sp.) on mushroom farms

Volume 19 Part 1 Article 19
Year 2016
Title: Sampling and detection of Dry Bubble (Lecanicillium sp.) on mushroom farms
Author: Gordon Rogers, Michael Kertesz, Warwick Gill, Judy Allan, Vicky Kobylski, Kasia Safianowicz, Lee Smith, Greg Seymour, John Doe, Miriam Jonson and Michael Jablonski

Major advances have been achieved in sampling for the detection of Lecanicillium. The key development is the use of a broad roller for detection of spores and mycelium over a large area (eg floor, wall), and then either extracting the inoculum as either elutions from the sampling roller, swabs from the roller, or direct transfer of the roller samples to agar “plates”.

Key achievements using the broad roller method include:
Lecanicillium detected in 90% of sites on a dry bubble-infected mushroom farms by sampling floors with large, short-napped paint roller sleeves using a combination of direct inoculation and swabbing
• The connection between dirty floors and crop infection strengthened by the detection of Lecanicillium on the soles of the shoes of harvester staff, supervisors and on the steps of picking trolleys.
Validation of the techniques is continuing on tray, shelf and bag farms, and the sampling techniques will be assessed for the detection of Cladobotryum and Trichoderma with a focus on roller sampling, touch plates and flies.

The diagnostics team have successfully developed PCR-based methods for the detection of Lecanicillium fungicola and generic Lecanicillium sp. The Lecanicillium fungicola probe is detecting L. fungicola with high accuracy (>90%) compared to conventional microbiological identification, and the Lecanicillium sp. probe is detecting a range of Lecanicillium spp. Amplicons are identified by sequencing. The Lecanicillium primers are part of a multiplex primer panel which is also detecting Trichoderma and Cladobotryum sp. The three fungal qPCR primers are working with accuracies in the high 90% range.

Methods to discriminate between alive and dead Lecanicillium sp. are being evaluated using a growth enrichment approach. Cultures are either heat-treated or not (control samples), incubated and quantified using qPCR. Live cells showed 43-fold increase in signal after incubation.

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