NGS tools aid screening for toxigenic Aspergilli in Pakistan.

Cafà G, Caggiano B, Bajwa B, Honey SF, Bhatti H, Buddie AG (2018).

State of the World’s Fungi Symposium, Royal Botanic Gardens, book of abstract P35

Aspergillus flavus

Poster presentation

Key chili and maize growing areas of the Punjab & Sindh province in Pakistan were selected for a focused baseline study to determine the levels of Aspergillus spp. that may lead to aflatoxin production.

Investigations were undertaken by a combination of classical (i.e. isolation and culture-based assays) and modern (i.e. molecular) techniques. Samples investigated included fresh chili, year-old corn cobs and soil samples from selected locations.

Aspergillus strains were obtained from corn cob but not from the fresh chilli samples. Isolates obtained from corn were screened for aflatoxin production using coconut milk agar test, with one strain giving a positive, fluorescent reaction.

Whole genome sequencing (WGS) with low coverage techniques were employed to screen the isolates for molecular differences in the ribosomal RNA gene cluster and mitochondrial genome, with the toxigenic strain proving to have a distinctive profile. Finally, the strains were subjected to MALDI-T0F-MS in order to obtain a proteomic fingerprint’ which was used to distinguish the toxigenic strain from the other isolates.

The NGS study was broadened to incorporate metabarcoding (using ITS and 16S) to determine the microbial biodiversity of the soil samples and screening for the presence of toxigenic strains. Using information gleaned from the WGS results, a putative toxigenic operational taxonomic unit (OTU) was observed in four of the 15 soil samples screened by metabarcoding. In conclusion, we present findings and recommendations for further research aimed at broadening the study and monitoring the fate of toxigenic Aspergillus strains in the field and in commodity storage facilities.