A PCR-based tool for the cultivation-independent monitoring of Pandora neoaphidis A. Fournier, J. Enkerli * , S. Keller, F. Widmer Molecular Ecology, Agroscope Reckenholz-Tänikon Research Station ART, Reckenholzstrasse 191, 8046 Zürich, Switzerland article info Article history: Received 18 December 2007 Accepted 20 March 2008 Available online 29 March 2008 Keywords: Entomopathogenic fungi Entomophthorales Erynia neoaphidis Aphid PCR-based detection in soil Winter survival Conservation biological control abstract Pandora neoaphidis is one of the most important fungal pathogens of aphids and has a great potential for use in biocontrol. Little is known on how this fungus persists in an area and in particular on its overwin- tering strategies. It is hypothesized that natural areas play an important role for survival and that soil may serve as a source of inoculum for new aphid populations in spring. To test these hypotheses, a cul- tivation-independent PCR-based diagnostic tool was developed, that allows the detection of P. neoaphidis in the environment. Two P. neoaphidis specific PCR primer pairs were designed, targeting sequences in the ribosomal RNA gene cluster. Specificity of both primer pairs was demonstrated with P. neoaphidis and non-target close entomophthoralean relatives. Moreover, single amplicons of expected sizes were obtained with both primer pairs from various environmental sample types, including aphid cadavers, plant material, and soil. The PCR-based diagnostic tool was applied to investigate the persistence of P. neoaphidis in soil samples obtained in 2004/2005 from a nettle field harboring infected aphids in fall 2004. P. neoaphidis was detected in every sample collected in November 2004 and March 2005, suggest- ing an overwintering stage of P. neoaphidis in top soil layers. The developed cultivation-independent PCR-based tool will be valuable for further investigation of the ecology of P. neoaphidis and for the development and future implementation of management strategies against aphids involving conserva- tion biocontrol. Ó 2008 Elsevier Inc. All rights reserved. 1. Introduction Pandora neoaphidis (Remaudière and Hennebert; Zygomycota, Entomophthorales) is one of the most important fungal pathogens of aphids (Hemiptera: Aphidoidea) in temperate areas (Pell et al., 2001; Steinkraus, 2006; Baverstock et al., 2008). This aphid-specific fungal pathogen has been reported to cause natural epizootics, which can dramatically reduce host populations, and thereby re- duce or prevent serious damage to crops (e.g. Keller and Suter, 1980; Feng et al., 1991; Steenberg and Eilenberg, 1995). Therefore, P. neoaphidis has a great potential for use in biological control of aphids. Two approaches, i.e. inundation and inoculation biocontrol, have been investigated in various studies, but have shown limited effectiveness (Powell and Pell, 2007). Conservation biocontrol, which is defined as ‘‘modification of the environment or existing practices to protect and enhance specific natural enemies or other organisms to reduce the effect of pests”(Eilenberg et al., 2001), is a promising third approach for the control of aphids with P. neoaphi- dis. However, for implementation of such an approach, profound knowledge on life cycle and ecology of the biocontrol organism is a prerequisite. Natural and semi-natural landscape elements such as field mar- gins, nettle patches, and natural meadows have been suggested to serve as reservoir for aphid-pathogenic entomophthoralean fungi (Keller and Suter, 1980; Barta and Cagan, 2003; Shah et al., 2004; Ekesi et al., 2005; Baverstock et al., 2008). It is hypothesized that after overwintering, new infection cycles are initiated in natural and semi-natural areas, from where fungal inoculum may dissem- inate to adjacent agricultural land and subsequently infect and control aphid populations developing in crops. However, knowledge on the winter survival strategies of entomophthoralean fungi and the following initiation of infection in spring is very limited. As most aphid species are holocyclic in cold temperate areas, like Switzerland (Keller and Suter, 1980; Blackman and Eastop, 2007), obligate aphid-pathogenic fungi such as P. neoaphidis may have to survive during winter in absence of larval or adult host stages, and/or to co-migrate with migratory alate aphids originating from other climatic zones (Feng et al., 2004, 2007). Some aphid-pathogenic entomophthoralean fungi have been reported to produce resting spores (zygospores or azy- gospores) in order to survive hostile conditions such as winter (Humber, 1989; Hajek and Wheeler, 1994). However, except for loricoconidia (Nielsen et al., 2003), which are thick-walled conidia no such survival structures have been reported for P. neoaphidis. Furthermore, the environment in which P. neoaphidis may over- winter is still not well defined. The fungus has been reported to 0022-2011/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.jip.2008.03.013 * Corresponding author. Fax: +41 0 44 377 72 01. E-mail address: juerg.enkerli@art.admin.ch (J. Enkerli). Journal of Invertebrate Pathology 99 (2008) 49–56 Contents lists available at ScienceDirect Journal of Invertebrate Pathology journal homepage: www.elsevier.com/locate/yjipa