MinION Sequencing of Fungi in Sub-Saharan African Air and a Novel LAMP Assay for Rapid Detection of the Tropical Phytopathogenic Genus Lasiodiplodia

Author:

King Kevin M.1,Canning Gail G. M.1,West Jonathan S.1ORCID

Affiliation:

1. Rothamsted Research, Harpenden AL5 2JQ, UK

Abstract

To date, there have been no DNA-based metabarcoding studies into airborne fungi in tropical Sub-Saharan Africa. In this initial study, 10 air samples were collected onto Vaseline-coated acrylic rods mounted on drones flown at heights of 15–50 meters above ground for 10–15 min at three sites in Ghana. Purified DNA was extracted from air samples, the internal transcribed spacer (ITS) region was amplified using fungal-specific primers, and MinION third-generation amplicon sequencing was undertaken with downstream bioinformatics analyses utilizing GAIA cloud-based software (at genus taxonomic level). Principal coordinate analyses based on Bray–Curtis beta diversity dissimilarity values found no clear evidence for the structuring of fungal air communities, nor were there significant differences in alpha diversity, based on geographic location (east vs. central Ghana), underlying vegetation type (cocoa vs. non-cocoa), or height above ground level (15–23 m vs. 25–50 m), and despite the short flight times (10–15 min), ~90 operational taxonomic units (OTUs) were identified in each sample. In Ghanaian air, fungal assemblages were skewed at the phylum taxonomic level towards the ascomycetes (53.7%) as opposed to basidiomycetes (24.6%); at the class level, the Dothideomycetes were predominant (29.8%) followed by the Agaricomycetes (21.8%). The most common fungal genus in Ghanaian air was cosmopolitan and globally ubiquitous Cladosporium (9.9% of reads). Interestingly, many fungal genera containing economically important phytopathogens of tropical crops were also identified in Ghanaian air, including Corynespora, Fusarium, and Lasiodiplodia. Consequently, a novel loop-mediated isothermal amplification (LAMP) assay, based on translation elongation factor-1α sequences, was developed and tested for rapid, sensitive, and specific detection of the fungal phytopathogenic genus Lasiodiplodia. Potential applications for improved tropical disease management are considered.

Funder

Morley Agricultural Foundation (UK) and Agritech Catalyst Round 8

UK Department for International Development (DFID) and the Global Challenges Research Fund

Biotechnology and Biological Sciences Research Council’s Industry Strategy Challenge Fund

UK-China Joint Centre for Sustainable Intensification in Agriculture (CSIA) and the Growing Health Institute Strategic Programme

Publisher

MDPI AG

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