ORIGINAL ARTICLE Molecular assays reveal the presence and diversity of genes encoding pea footrot pathogenicity determinants in Nectria haematococca and in agricultural soils E. Etebu and A.M. Osborn Department of Animal and Plant Sciences, The University of Sheffield, Sheffield, UK Introduction Peas (Pisum sativum) are grown in over 87 countries worldwide (McPhee 2003) providing food for humans and feed for domestic animals (Hargrove 1986; Hulse 1994; Patriarca et al. 2002). Total global production has increased considerably over the years with production now amounting to 10Æ5 and 7 million tonnes of dry and fresh peas, respectively (Duke 1981; FAO 1994, 2001). Peas have important nutritional qualities and are considered to be the predominant export crop in world trade of pulses, representing 40% of the latter (Oram and Agcaoili 1994). Root rot (footrot) disease has been identified as a major constraint on pea production (Graham and Vance 2003), with infections occurring worldwide (Hagedorn 1976; Persson et al. 1997). Infection in peas begins at the cotyledonary attachment area, below ground epicotyl, hypocotyl and upper tap root, and its symptoms are char- acterized by a reddish brown to black discolouration of the root system around the area of cotyledon attachment. Above ground symptoms are characterized by stunted growth and yellowing and necrosis of foliage (Hagedorn 1991; Kraft and Boge 2001). Root rot limits the uptake of water and nutrients, causes early stagnation of root growth, and adversely affects symbiotic nitrogen fixation (Tu 1987). In peas, the disease is caused by a number of fungi, but most notably by Nectria haematococca (ana- morph Fusarium solani f. sp. pisi) (Funnell et al. 2001). Nectria haematococca is pathogenic on all commercial processing pea cultivars (Hagedorn 1991; Gru ¨nwald et al. Keywords footrot disease in peas, Fusarium solani, Nectria haematococca, pathogenicity genes (PEP, PDA), soil DNA. Correspondence Mark Osborn, Department of Animal and Plant Sciences, The University of Sheffield, Western Bank, Sheffield S10 2TN, UK. E-mail: a.m.osborn@sheffield.ac.uk 2008 ⁄ 1208: received 14 July 2008, revised and accepted 20 October 2008 doi:10.1111/j.1365-2672.2008.04130.x Abstract Aim: The aim of this study was to develop molecular assays for investigating the presence and diversity of pathogenicity genes from the pea footrot patho- gen Nectria haematococca (anamorph Fusarium solani f.sp. pisi) in soils. Methods and Results: Polymerase chain reaction (PCR) assays were developed to amplify four N. haematococca pathogenicity genes (PDA, PEP1, PEP3 and PEP5) from isolates and soil-DNA from five agricultural fields with a prior foo- trot history. A collection of 15 fungi isolated on medium selective for Fusarium spp. exhibited variation in their virulence to peas as assessed via a disease index (DI: 0–5; no virulence to the highest virulence). PCR analyses showed that three isolates in which all four pathogenicity genes were detected resulted in the highest DI (>3Æ88). All four pathogenicity genes were detected in soil-DNA obtained from all five fields with a footrot disease history, but were not ampli- fied from soils, which had no footrot history. Denaturing gradient gel electro- phoresis and ⁄ or sequence analysis revealed diversity amongst the pathogenicity genes. Conclusion: The PCR assays developed herein enable the specific detection of pathogenic N. haematococca in soils without recourse to culture. Significance and Impact of the Study: Molecular assays that specifically target pathogenicity genes have the capacity to assess the presence of the footrot- causing pathogen in agricultural soils. Journal of Applied Microbiology ISSN 1364-5072 ª 2009 The Authors Journal compilation ª 2009 The Society for Applied Microbiology, Journal of Applied Microbiology 106 (2009) 1629–1639 1629