Occurrence and diversity of free-living protozoa on butterhead lettuce

The occurrence and diversity of free-living protozoa (FLP) on butterhead lettuce (Lactuca sativa L.) was investigated using four different sampling techniques (washing, swabbing, homogenization, and excising). FLP were recovered from all leaf samples (n = 64), and cultures were FLP-positive after 1 week. Identification of FLP was performed by light microscopy and sequencing of denaturing gradient gel electrophoresis (DGGE)-separated 18S rRNA gene fragments. Bodo saltans, Spumella (-like) spp. and Cercozoa were the most common heterotrophic nanoflagellates. Amoebae belonged mainly to the Vannellida and Tubulinida. Colpoda steinii and Cyclidium glaucoma were the most common ciliates. The total number of FLP on middle leaves estimated by the Most Probable Number method ranged from 9.3 × 102 MPN/g to 2.4 × 105 MPN/g leaf, with flagellates (92 MPN/g to 2.4 × 105 MPN/g) being more abundant than amoebae (< 3 MPN/g to 9.3 × 103 MPN/g) and ciliates (< 3 MPN/g to 9.3 × 102 MPN/g). Washing or rinsing leaves followed by spin-drying in a household salad spinner reduced the protozoan number with maximum one log unit. Our survey shows that FLP on lettuce leaves are a common and diverse but largely unexplored group of microorganisms.

Research highlights
► Free-living protozoa (FLP) are present on lettuce leaves and ready-to-eat lettuce.
► The FLP diversity (amoebae, flagellates, and ciliates) on lettuce leaves is high.
► Combine microscopy and DNA sequencing to determine the FLP diversity.
► Washing leaves followed by spin-drying scarcely reduced the FLP number.