MinION sequencing of fungi in Sub-Saharan African air, and a novel LAMP assay for rapid detection of the tropical phytopathogenic genus Lasiodiplodia

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 metres above ground for 10-15 minutes 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 Co-Ordinate analyses based on Bray-Curtis beta diversity dissimilarity values found no clear evidence for structuring of fungal air communities 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). In Ghanaian air samples, despite the very short flight times, ~90 operational taxonomic units (OTUs) were identified in each sample, with no statistical differences in alpha diversity between air samples from different locations, vegetation types or height above ground. 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 Dothideomyectes 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. The potential applications for improved tropical disease management are considered.