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PCR Primers with Enhanced Specificity for Nematode-Trapping Fungi (Orbiliales)

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Abstract

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.

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Acknowledgements

The authors thank Ole Becker and James Borneman for providing cultures of Brachyphoris oviparasitica for 28s sequencing. David M. Rizzo generously provided laboratory space and equipment. UC Bodega Marine Laboratory and Reserve provided a travel grant for B. Jaffee. Antonio Izzo provided valuable advice during primer development. We thank Donald Pfister and two anonymous reviewers for useful comments on previous drafts.

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Authors and Affiliations

  1. Farlow Herbarium and Department of Organismic and Evolutionary Biology, Harvard University, 22 Divinity Avenue, Cambridge, MA, 02138, USA

    Matthew E. Smith

  2. Department of Nematology, University of California at Davis, One Shields Avenue, Davis, CA, 95616, USA

    Matthew E. Smith & Bruce A. Jaffee

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Correspondence to Matthew E. Smith.

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Smith, M.E., Jaffee, B.A. PCR Primers with Enhanced Specificity for Nematode-Trapping Fungi (Orbiliales). Microb Ecol 58, 117–128 (2009). https://doi.org/10.1007/s00248-008-9453-0

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