Virulence, sporulation, and elicitin production in three clonal lineages of Phytophthora ramorum D.K. Manter a, * , E.H. Kolodny a , E.M. Hansen b , J.L. Parke c a USDA-ARS, Soil-Plant-Nutrient Research, 2150 Centre Ave., Bldg. D, Suite 100, Fort Collins, CO 80526, USA b Botany and Plant Pathology, Oregon State University, Corvallis, USA c Department of Crop and Soil Science, Oregon State University, Corvallis, USA article info Article history: Accepted 27 April 2010 Keywords: Sudden oak death Plant disease Rhododendron qPCR ELISA abstract Phytophthora ramorum populations are clonal and consist of three lineages. Recent studies have shown that the clonal lineages may have varying degrees of aggressiveness on some host species, such as Quercus rubra. In this study, we examined virulence, sporulation and elicitin production of five P. ramorum isolates from each of the three clonal lineages. Virulence (lesion size) and sporulation (sporangia production) were determined on wound-inoculated detached leaves of Rhododendron cata- wbiense ‘Nova Zembla’. Lesion area differed between the clonal lineages (p < 0.001) with the EU1 and NA2 isolates producing significantly greater lesion areas than did NA1 isolates on inoculated leaves (approx. 4.2, 3.6, and 0.8 cm 2 respectively). Similarly, lineages EU1 and NA2 produced significantly more sporangia per leaf (p < 0.001) than did lineage NA1 (approx. 800, 1000, and 300 sporangia per leaf respectively). Real-time PCR assays detected expression of the class I elicitins (ram-a1 and ram-a2) in all 15 isolates. Of the two elicitins, only the ram-a2 differed between lineage (p < 0.0001) with nearly 2-fold higher levels of expression in the EU1 and NA2 lineages as compared to the NA1 lineage. Ram-a2 expression showed a positive linear relationship with isolate virulence or lesion size (R 2 ¼ 0.707). A significant, positive, linear relationship was also observed between ram-a2 expression and sporulation although it was not as strong (R 2 ¼ 0.209). In summary, isolates belonging to clonal lineages EU1 and NA2 are generally more virulent, produce more sporangia, and produce more ram-a2 elicitin in vitro than isolates belonging to lineage NA1. Published by Elsevier Ltd. 1. Introduction Sudden Oak Death caused by the plant pathogenic oomycete, Phytophthora ramorum, contributes to significant mortality in various oak species from central coastal California to southern Oregon [1]. Currently, three clonal lineages of P. ramorum have been identified in the United States [2]; the North American lineage (lineage NA1, mating type A2) is responsible for infections in CA and OR forests [3]. The European lineage (lineage EU1, predominantly A1) is responsible for infections in Europe, but has also been found in nurseries in OR and WA [4]. A third lineage (NA2, mating type A2) has only been isolated in a few instances from nurseries in WA and CA [5]. Phenotypic and adaptive differences have been observed between NA1 and EU1 isolates of P. ramorum [6e8]. NA1 isolates were slower growing, and varied more between isolates, than their EU1 counterparts [7]. Within the NA1 isolates, Brasier et al. [7] noted two general colony morphologies, wild type (fast-growing) and non-wild type (typically slow-growing). Interestingly, subcul- tures from individual NA1 isolates gave rise to both the wild type and non-wild type morphologies, although the factors responsible for this variability are unknown. Virulence of the EU1 and NA1 isolates was also examined by phloem inoculations of mature Quercus rubra stems, a rigorous test of comparative pathogenic ability. In three different experiments, Brasier et al. [7] found that populations of EU1 isolates produced greater lesion areas as compared to populations of NA1 isolates. The lower virulence in the NA1 isolates, as compared to the EU1 isolates, was present for both the wild type and non-wild type NA1 morphologies, although the slow-growing non-wild type cultures were the least virulent. Elicitins are 10 kDa proteins produced by most Phytophthora and Pythium spp. and are thought to aid in sterol uptake from the environment, an absolute requirement for sporulation [9]. Phy- tophthora spp. can discriminate between sterols and differentially utilize sterols from their growth media, affecting growth and sexual reproduction [10,11]. In addition, Phytophthora elicitin production * Corresponding author. Tel.: þ1 970 492 7255; fax: þ1 970 492 7213. E-mail address: daniel.manter@ars.usda.gov (D.K. Manter). Contents lists available at ScienceDirect Physiological and Molecular Plant Pathology journal homepage: www.elsevier.com/locate/pmpp 0885-5765/$ e see front matter Published by Elsevier Ltd. doi:10.1016/j.pmpp.2010.04.008 Physiological and Molecular Plant Pathology 74 (2010) 317e322