Measuring diversity of endophytic fungi in leaf fragments: does size matter?

Endophytic fungi inhabit living plant tissues without causing disease symptoms. Although abundant, the extent of their contribution to fungal biodiversity remains unclear. Since endophytic fungi are poorly known, especially in the tropics, current estimates of fungal species are probably conservative. Here we tested strategies for sampling endophytic fungi in tropical plants. We compared the number of fungi isolated from 400 mm2 leaf pieces that were divided into increasingly small fragments. Leaf pieces were surface-sterilized, cut into fragments and plated on culture media. For a given area, cutting leaf pieces into smaller fragments significantly increased the number of fungal morphospecies recovered. There was a strong linear relationship between size of fragments and number of fungi isolated. By extrapolation, an estimated 16 +/- 3 fungi could be recovered from a 2 x 2 cm leaf piece, using infinitely small fragments. This represents a large part of the fungal diversity estimated to exist in leaf endophytes in a population. We conclude that reducing the size and increasing the number of leaf fragments will increase the number of fungal species isolated. This strategy will help to estimate real values of endophytic fungal diversity.