Diplanetism and microcyclic sporulation in Phytophthora ramorum By E. Moralejo and E. Descals Institut Mediterrani dÕEstudis Avançats, IMEDEA (CSIC-UIB), Miquel Marque ` s 21, 07190 Esporles, Balearic Islands, Spain. E-mail: vieaemr@uib.es (for correspondence) Summary The zoosporic phase of the pathogen Phytophthora ramorum plays a crucial role in the process of plant infection, yet little is known about the fate of zoospores failing to target their hosts. Here, we describe new stages in the life cycle of P. ramorum concerning the in vitro development of monomorphic diplanetism and microcyclic sporulation in free water. Papillate cysts were formed after zoospore suspensions of isolates of the EU1 and NA1 clonal lineages were vortexed. Cysts usually germinated directly forming an emerging tube, or indirectly by releasing a secondary zoospore, which leaves behind an empty cyst with a short evacuation tube. Germinate cysts frequently developed either an appressorium or a microsporangium both terminally. We also observed microcyclic sporulation, i.e. sporangia indirectly germinated by forming a microsporangium, as in microcyclic conidiation of true fungi. Temporal progress of encysted zoospores in solution showed that percentage of germination varied significantly among and within isolates as well as between experiments, suggesting that germination is partly ruled by internal mechanisms. Diplanetism and microcyclic sporulation in P. ramorum may provide a second opportunity for host infection and may increase the chance of long dispersal in moving water. 1. Introduction The zoosporic phase is of much importance in the infection process, survival and dispersal of plant pathogenic oomycetes (Hickman 1970; Walker and van West 2006; Jeger and Pautasso 2008); yet, what happens to zoospores released from sporangia and failing to target their host remains poorly known (Deacon and Donaldson 1993). Zoospores have a limited capacity for self-dispersal in water films but they can be passively dispersed over long distances by moving water (Newhook et al. 1981). In some species of the genera Phytophthora and Pythium, zoospores swim until they encyst permanently or they may display a first swimming period followed by encystment, emergence and a second swimming period followed by encystment, a phenomenon named diplanetism (Drechsler 1930; Dick 2001). Because the emergent secondary zoospore does not differ morphologically from the primary one, the term monomorphic diplanetism has been coined to distinguish it from that of the dimorphic zoospores in the Saprolegniaceae (see Blackwell 1949; Dick 2001). In 1930, Drechsler reviewed the literature concerning the occurrence of Ôrepetitional diplanetismÕ in the genus Phytophthora. A full description accompanied by drawings of the different stages of diplanetism in Phytophthora was provided. Some observations by other researchers (Murphy 1920; Sawada 1927) were pointed out by Drechsler, such as the diversity of fungal structures derived from the germination of zoospore cysts and the apparent increment in biomass of mycelia from single germinated zoospores (Sawada 1927). Since then, despite the recognition of the importance of diplanetism by modern Phytophthora researchers (e.g. Walker and van West 2006), the role of zoospores in the epidemiology of Phytophthora diseases has been seldom investigated (Jeger and Pautasso 2008). In the few notable exceptions, however, studies of zoospore taxis and of genes involved in pathogenesis at the zoospore stage have shown a great refinement in zoospore adaptation to the environment (Deacon and Donaldson 1993; von Broembsen and Deacon 1996; or see reviews by Judelson and Blanco 2005 and Walker and van West 2006). In the course of in vitro inoculation experiments with Phytophthora ramorum, we observed many empty cysts in resting zoospore solutions, each exhibiting a wide operculum through which supposedly a zoospore had been released, such as those described previously in other Phytophthoras (e.g. Drechsler 1930; Blackwell 1949; Ho and Hickman 1967; Hemmes and Holh 1971; Ho and Zentmyer 1977). The exotic oomycete P. ramorum is a multiple-host pathogen recently introduced in North America and Europe that has become notorious for causing a huge mortality of native oaks and tan oaks along the Pacific coast of the USA, where the disease is known as Sudden Oak Death (SOD; Rizzo et al. 2005). It also infects several ornamental plants in both continents, where the plant trade has served as an important pathway for long distance dispersal of the pathogen (Rizzo et al. 2005; Moralejo et al. 2009b; Xu et al. 2009). Research on P. ramorum has mainly focused on elucidating different epidemiological aspects such as those related to the pathogenÕs population structure, inoculum source, breeding system and potential host range (e.g. Brasier and Kirk 2004; Davidson et al. 2005, 2008; Denman et al. 2005; Tooley and Kyde 2007; Moralejo et al. 2009a). Yet, few studies have dealt with basic research on the pathogenÕs life cycle, despite this being essential for understanding the epidemiology and ecology of SOD (Moralejo et al. 2006a; Davidson et al. 2008). In this article, we describe different fungus structures derived from zoospore suspensions of P. ramorum, and we show how the proportion of these structures evolves over time in vitro. We aim to characterize the sequence of stages from zoospore encystment to the formation of secondary zoospores and microsporangia under controlled conditions simulating aquatic environments, and elucidating whether the secondary zoospores are capable of infection as well. Finally, the relevance that such structures might have in the ecology of P. ramorum is highlighted. For. Path. 41 (2011) 349–354 doi: 10.1111/j.1439-0329.2010.00674.x Ó 2010 Blackwell Verlag GmbH Received: 12.3.2010; accepted: 20.5.2010; editor: L. Belbahri http://wileyonlinelibrary.com/