High-throughput culturing of fungi from plant litter by a dilution-to-extinction technique

Abstract

High-throughput bacterial cultivation has improved the recovery of slow-growing and previously uncultured bacteria. The most robust high-throughput methods are based on techniques of 'dilution to extinction' or 'extinction culturing'. The low-density partitioning of CFUs in tubes or microwells exploits the fact that the number of culturable species typically increases as inoculum density decreases. Bacterial high-throughput culturing methods were adapted to fungi to generate large numbers of fungal extinction cultures. The efficiency of extinction culturing was assessed by comparing it with particle filtration and automated plate-streaking. Equal volumes of particle suspension from five litter collections of the New Zealand forest tree Elaeocarpus dentatus were compared. Dilute particle suspensions of litter were pipetted into 48-well tissue culture plates containing 1 mL of agar medium per well. Particle volumes from the same samples were applied to continuous agar surfaces in Omnitray plates by automated streaking, and fungal diversity and richness were measured. The spectrum of isolates was assessed by microscopy and sequencing of the ITS or 28S region of the rRNA gene. Estimates of species diversity between the two methods were comparable, but extinction culturing increased species richness. Compared with plating methods using continuous surfaces, extinction culturing distributes fungal propagules over partitioned surfaces. Intercolony interactions are reduced, permitting longer incubation times, and colony initiation and recovery improved. Effort to evaluate and recover colonies from fungal isolation plates was substantially reduced.