PCR primers with enhanced specificity for nematode-trapping fungi (Orbiliales).

Nematode-trapping fungi, a monophyletic lineage within the Orbiliales (Ascomycota), use specialized structures to capture and consume nematodes in soil, leaf litter, and other substrates. These fungi have been studied both because of their unique predatory life history and because they are potential control agents of important plant- and animal-parasitic nematodes. Ecological studies of nematode-trapping fungi have primarily used culture-based methods, but molecular detection techniques are now available and should be useful for studying this group. We developed Orbiliales-specific PCR primers for the ITS and 28s rDNA to directly detect nematode-trapping fungi without culturing and also to screen fungal isolates for phylogenetic placement in the Orbiliales. We used these primers to selectively amplify, clone, and sequence Orbiliales DNA extracted from soil, litter, and wood, and we compared the results of molecular detection with those obtained using a culture-based method. Of the eight species of nematode-trapping Orbiliales detected with the culture-based assay, only three were detected with PCR. The molecular assay, however, detected 18 species of uncultured Orbiliales, many of which are closely related to nematode-trapping fungi and fungal parasites of nematode eggs. Our results suggest that the combined use of Orbiliales-specific primers and culture-based techniques may benefit future studies of nematophagous fungi.

Response of nematode-trapping fungi to organic substrates in a coastal grassland soil.

To understand why Arthrobotrys oligospora and other nematode-trapping fungi are common and sometimes abundant in the coastal grassland soils of the Bodega Marine Reserve (BMR, Sonoma County, CA), we examined how resident trapping fungi responded to the addition of eight organic substrates (lupine leaves, grass leaves, dead isopods, dead moth larvae, isopod faeces, deer faeces, shrimp shells, and powdered chitin). We were especially interested in the effects of dead isopods because isopods are abundant at BMR and because previous studies had documented strong responses of A. oligospora to other arthropods (dead moth larvae). Soil from BMR was packed into vials (40 g dry mass equivalent per vial with water potential at -230 kPa and bulk density at 0.9 gcm(-3)), and one substrate or no substrate was added to the soil surface. After 30 d at 20 degrees C, trapping fungi were quantified by dilution plating and most probable number procedures. The response of A. oligospora was inversely related to substrate carbon:nitrogen (C:N) ratio: substrates with low C:N ratios (dead isopods, lupine leaves, dead moth larvae) usually caused large increases in A. oligospora whereas those with higher C:N ratios (isopod faeces, deer faeces, grass leaves) did not. An exception was chitin powder, which had a low C:N ratio, but which did not cause A. oligospora to proliferate. Responses of A. oligospora were directly related to the quantity of nitrogen added with each substrate, and those substrates that caused large increases in resident nematodes usually caused large increases in A. oligospora. Other trapping fungi did not respond as strongly as A. oligospora.