·PSSPM! doi:10.5598/imafungus.2017.08.02.09 8(#('*$"*1'" Phytophthora ARTICLE Xiao Yang1, Brett M. Tyler2, and Chuanxue Hong1 1Hampton Roads Agricultural Research and Extension Center, Virginia Tech, Virginia Beach, VA 23455, USA; corresponding author e-mail: [email protected] 2Center for Genome Research and Biocomputing, and Department of Botany and Plant Pathology, Oregon State University, Corvallis, OR 97331, USA )#A comprehensive phylogeny representing 142 described and 43 provisionally named Phytophthora species 3*4 is reported here for this rapidly expanding genus. This phylogeny features signature sequences of 114 ex-types and oomycetes numerous authentic isolates that were designated as representative isolates by the originators of the respective species. systematics Multiple new subclades were assigned in clades 2, 6, 7, and 9. A single species P. lilii was placed basal to clades 1 to taxonomy 5, and 7. Phytophthora stricta was placed basal to other clade 8 species, P. asparagi to clade 6 and P. intercalaris to evolution clade 10. On the basis of this phylogeny and ancestral state reconstructions, new hypotheses were proposed for the plant pathology evolutionary history of sporangial papillation of Phytophthora species. Non-papillate ancestral Phytophthora species were inferred to evolve through separate evolutionary paths to either papillate or semi-papillate species. $1 Submitted: 8 June 2017; Accepted: 31 October 2017; Published: 21 November 2017. INTRODUCTION A sound taxonomic system is foundational for correctly identifying Phytophthora species and safeguarding The genus Phytophthora has had profound impacts on agriculture, forestry, and natural ecosystems. Traditionally, human history by causing agriculturally and ecologically taxonomy of the genus was based on morphological important plant diseases (Erwin & Ribeiro 1996). Among OHBDBO@GD?& [=LVB>GL@BX ><DMH<X<ZWFB?GV OXB??E[OB@E<L the most notorious Phytophthora species is P. infestans, of Phytophthora species was established by Waterhouse cause of the late blight disease, which was the primary IH<OXB??E[GV@HG?MGOEG?EL@<?EZD<=M?FB?GV<L@HG cause of the Irish potato famine from 1845 to 1852 in which ><DMH<X<ZW<[?M<DBLZEBH<><@HBXXE?>BLVO<L[Z=DB@E<L<[ approximately one million people died and 1.5 million antheridia. However, plasticity in morphological characters emigrated from Ireland (Turner 2005). Another example B><LZ?@ E?<XB@G? <[ ELVEEV=BX ?MGOEG? E? ?EZLE[OBL@ ?< is the sudden oak death pathogen, P. ramorum, that has is homology or homoplasy among different species. For killed millions of coast live oak, tanoak and Japanese larch example, isolates of P. constricta (Rea et al. 2011), P. trees, and has permanently altered the forest ecosystems gibbosa (Jung et al. 2011), P. lateralis (Kroon et al. 2012), in California and Oregon, USA (Goheen et al. 2002, Rizzo P. mississippiae (Yang et al. 2013), and P. multivesiculata et al. 2002, Rizzo et al. 2005). Other species, such as P. (Ilieva et al. 1998) all produce a mixture of semi-papillate cinnamomi, P. nicotianae, and P. sojae, can also cause and non-papillate sporangia. Many non-papillate species highly destructive plant diseases (Erwin & Ribeiro 1996). recovered from irrigation water such as Phytophthora The impact caused by Phytophthora species has continued hydropathica (Hong et al. 2010) and P. irrigata (Hong et al. to increase with the emergence of new pathogens and 2008) were morphologically inseparable from P. drechsleri, diseases. The number of species known in the genus has while sequence analyses demonstrated that they are distinct doubled during the past decade due to extensive surveys in species. Also, production of many morphological structures previously unexplored ecosystems such as natural forests BLV MHW?E<X<ZEOBX [GB@=DG? LGGV? ?MGOE[O GLED<L>GL@BX (Jung et al. 2011, 2017, Rea et al. 2010, Reeser et al. 2013, conditions, while observation of these features requires Vettraino et al. 2011), streams (Bezuidenhout et al. 2010, ?=F?@BL@EBX @DBELELZ BLV GMGD@E?G *E[[O=X@W EL <F@BELELZ Brazee et al. 2017, Reeser et al. 2007, Yang et al. 2016), important morphological data can impair accurate species riparian ecosystems (Brasier et al. 2003a, 2004, Hansen EVGL@E[OB@E<L et al. 2012), and irrigation systems (Hong et al. 2010, With the advent of DNA sequencing, the taxonomic 2012, Yang et al. 2014a, b). The total number of formally concept for the genus has evolved from morphology to named species in the genus was about 58 in 1996 (Erwin & molecular phylogeny-based (Blair et al. 2008, Cooke et al. Ribeiro 1996), but now is more than 150. In addition, some 2000, Kroon et al. 2004, Lara & Belbahri 2011, Martin et provisionally or informally named species are also expected al. 2014, Martin & Tooley 2003, Robideau et al. 2011, Villa to be formally described in the near future. et al. 2006). In particular, the availability of whole genome © 2017 International Mycological Association You are free to share - to copy, distribute and transmit the work, under the following conditions: Attribution: 7<=>=?@B@@DEF=@G@HGI<DJEL@HG>BLLGD?MGOE[GVFW@HGB=@H<D<DXEOGL?<DF=@L<@ELBLWIBW@HB@?=ZZG?@?@HB@@HGWGLV<D?GW<=<DW<=D=?G<[@HGI<DJ Non-commercial: 7<=>BWL<@=?G@HE?I<DJ[<DO<>>GDOEBXM=DM<?G? No derivative works: 7<=>BWL<@BX@GD@DBL?[<D><DF=EXV=M<L@HE?I<DJ For any reuse or distribution, you must make clear to others the license terms of this work, which can be found at http://creativecommons.org/licenses/by-nc-nd/3.0/legalcode. Any of the above conditions can be waived if you get permission from the copyright holder. Nothing in this license impairs or restricts the author’s moral rights. VOLUME 8 · NO. 2 355 ?#" et al. sequences from P. sojae, P. ramorum (Tyler et al. 2006) and that were designated as representative isolates by the P. infestans (Haas et al GLBFXGV@HGEVGL@E[OB@E<L<[ originators of the respective species names (Table 1). The genetic markers useful for multi-locus phylogenies (Blair et majority of these isolates were provided by the originators of al. 2008). the respective species, while the rest were purchased from Cooke et al VGGX<MGV @HG [D?@ ><XGO=XBD the Westerdijk Fungal Biodiversity Institute (CBS), Utrecht, ARTICLE phylogeny for the genus by analyzing sequences of the The Netherlands. internal transcribed spacer region (ITS) of 51 species. Kroon et al. (2004) constructed a phylogeny based on sequences =8# of four nuclear and mitochondrial genes of 48 species, and To extract genomic DNA (gDNA), an approximately 5 × 5 Blair et al. (2008) produced a sophisticated phylogeny based mm culture plug of each isolate was taken from the actively on sequences of seven nuclear genetic markers. That multi- growing area of a fresh culture. This was then grown in 20 % locus phylogeny divided 82 Phytophthora species into 10 OXBDE[GV­FD<@HXE>BFGBLFD<@H[<DZD<IELZBP. infestans phylogenetically well-supported clades. Martin et al. (2014) isolate 27A8) at room temperature (ca. 23 °C) for 7–14 d analyzed sequences of seven nuclear and four mitochondrial to produce a mycelial mass. The mass was then blot-dried genes of 90 formally named and 17 provisional species and using sterile tissue paper and then lysed in liquid nitrogen provided phylogenies including 10 clades, almost identical to or using a FastPrep®-24 system (MP Biomedicals, Santa that of Blair et al. (2008), except that P. quercina and P. sp. Ana, CA). gDNA was extracted using the DNeasy® Plant Mini ohioensis were excluded from clade 4 and grouped into a kit (Qiagen, Valencia, CA) or the Maxwell® Plant DNA kit in potentially new clade. combination with a Maxwell® Rapid Sample Concentrator A comprehensive molecular phylogeny is required (Promega, Madison, WI). to understanding the evolution of Phytophthora species. Although discordance has been found between the molecular =#%($##@" phylogeny and the morphology-based taxonomy (Cooke et al. A set of primers for seven genetic markers were used for 2000, Ersek & Ribeiro 2010), correlations have been observed *& B>MXE[OB@E<L ELOX=VELZ ©EF<?<>BX MD<@GEL between molecular phylogenies and individual morphological FG@B@=F=XEL$@=FGX<LZB@E<L[BO@<D BXMHB) and physiological traits. Recent studies indicated that enolase (Enl), heat shock protein 90 (HSP90), 28S ribosomal species in individual clades or subclades are mostly identical DNA (28S), and tigA gene fusion protein (TigA) as indicated in sporangial papillation, and optimum and maximum growth in Blair et al. (2008). PCR reaction mixtures were prepared temperatures (Cooke et al. 2000, Kroon et al. 2012, Martin with the Takara Taq DNA polymerase (Takara Shuzo, Shiga, et al. 2012, Yang 2014). However, there was limited to no Japan) according to the manufacturer’s instructions. The correlation between phylogeny and the morphology of PCR cycling protocol was the same as indicated by Blair ?G=BX <DZBL? ?=OH B? BL@HGDEVEBX O<L[Z=DB@E<L (<<JG et et al. (2008), except that the Eppendorf® Mastercycler® Pro al. 2000, Kroon et al. 2012, Martin et al. 2012, Yang 2014). thermal cycler (Eppendorf, Hamburg) was used in this study. These studies have implied that divergence in sporangial &XX (©MD<V=O@?IGDGGBX=B@GV[<D?=OOG??[=XB>MXE[OB@E<L morphology and variation in environmental specialization using agarose gel electrophoresis. Unsuccessful PCR may be the keys in the evolutionary history of Phytophthora B>MXE[OB@E<L? IGDG DGMGB@GV =?ELZ B ><VE[GV MD<@<O<X @< species. Nevertheless, these hypotheses need to be further B@@G>M@ ?=OOG??[=X B>MXE[OB@E<L? FW <M@E>EELZ BLLGBXELZ tested and the exact evolutionary history of the genus temperature using gradient PCR (typically with lower Phytophthora warranted more investigation. annealing temperatures) or using the GoTaq® Flexi DNA In this study, an expanded phylogeny, including more Polymerase (Promega, Madison, WI) PCR mixture system.