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A rapid and miniaturized system using Alamar blue to assess fungal spore viability: implications for biosecurity

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Abstract

Long-lasting viable fungal spores are one of the important aspects in emergence, spread and disease development of pathogenic fungi. We developed a rapid and miniaturized system using Alamar Blue (resazurin dye; 7-hydroxy-3H-phenoxazin-3-one 10-oxide) for assessing fungal spore viability, using the ascomycete Leptosphaeria maculans (causing blackleg disease on canola) as a ‘model pathogen’. The assay is dependent on the metabolic activity of viable fungal spores to convert the dark blue of resazurin (maximum absorbance 605 nm) into the pink colour of resorufin (maximum absorbance 573 nm). The Alamar Blue assay uses an optimised micro-titre based format that was far superior for determining fungal spore viability in comparison with current conventional techniques including trypan blue staining, a TC10 cellometer cell counter, or by assessing germination of the spores under the microscope. This new assay was also more rapid and reproducible than current conventional tests to detect viable spores. Viable spores could be reliably detected within two hours. The successful application of the Alamar Blue assay to measure fungal spore viability in the current study has important benefits for biosecurity operations relating to faster and more reliable confirmation of viability of potential invasive exotic fungal pathogens and in minimising any consequent disease outbreaks. The effectiveness of the Alamar Blue assay was confirmed by successfully determining the relative retention times of viable L. maculans ascospores across a range of different potential spore-carrier materials, including steel, fabric, wood, paper, rubber and leather, over a time period of eight months. To further confirm the wide applicability of the Alamar Blue assay, it was successfully applied to detect viable spores of fungal pathogens of diverse taxonomic groups, including Kabatiella caulivora, Magnaporthe oryzae and Puccinia striiformis f.sp. tritici, and also of the yeast Saccharomyces cerevisiae.

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Acknowledgments

We greatly appreciate the scholarship and operating funding provided by the Plant Biosecurity CRC for project CRC62042: ‘Curtailing and managing exotic fungal spore incursion into Australia’, and financial support by School of Plant Biology at the University of Western Australia. We thank Mr. Robert Creasy and Dr. Michael Considine from the University of Western Australia for the use of facilities and technical help during these studies.

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Authors and Affiliations

  1. School of Agriculture and Environment and the UWA Institute of Agriculture, Faculty of Science, The University of Western Australia, 35 Stirling Highway, Crawley, WA, 6009, Australia

    Papori Barua, Ming Pei You & Martin J. Barbetti

  2. School of Veterinary and Life Sciences, Murdoch University, South Street, WA, 6105, Australia

    Kirsty Bayliss

  3. Department of Agriculture and Food Western Australia, 3 Baron-Hay Court, South Perth, WA, 6151, Australia

    Vincent Lanoiselet

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  1. Papori Barua
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Correspondence to Martin J. Barbetti.

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Barua, P., You, M.P., Bayliss, K. et al. A rapid and miniaturized system using Alamar blue to assess fungal spore viability: implications for biosecurity. Eur J Plant Pathol 148, 139–150 (2017). https://doi.org/10.1007/s10658-016-1077-5

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