Microbial Ecology

Diversity of Peronosporomycete (Oomycete) Communities Associated with the Rhizosphere of Different Plant Species Jessica M. Arcate, Mary Ann Karp and Eric B. Nelson

Department of Plant Pathology, Cornell University, 334 Plant Science Building, Ithaca, NY 14853, USA

Received: 15 September 2004 / Accepted: 12 January 2005 / Online Publication: 3 January 2006

Abstract Introduction Peronosporomycete (oomycete) communities inhabiting The Peronosporomycetes are a large, ecologically, and the rhizospheres of three plant species were characterized phylogenetically distinct group of eukaryotes found most and compared to determine whether communities commonly in terrestrial and aquatic habitats. They obtained by direct soil DNA extractions (soil communi- include well-known genera of plant pathogens such as ties) differ from those obtained using baiting techniques Aphanomyces, Peronospora, Phytophthora, and Pythium, (bait communities). Using two sets of Peronosporomy- most of which are soil-borne and infect subterranean cete-specific primers, a portion of the 50 region of the plant parts such as seeds, roots, and hypocotyls. This large subunit (28S) rRNA gene was amplified from DNA group also includes other important genera such as extracted either directly from rhizosphere soil or from Saprolegnia, Achlya, and Lagenidium, which are patho- hempseed baits floated for 48 h over rhizosphere soil. genic to fish, insects, crustaceans, and mammals [17]. Amplicons were cloned, sequenced, and then subjected Although these organisms have received much attention to phylogenetic and diversity analyses. Both soil and bait in terms of the diseases they cause, few other details of communities arising from DNA amplified with a Per- their ecology are known. onosporomycetidae-biased primer set (Oom1) were For many years, Peronosporomycetes were believed dominated by Pythium species. In contrast, communities to be closely related to fungi. It was assumed, therefore, arising from DNA amplified with a Saprolegniomyceti- that they shared similar ecological traits. However, it is dae-biased primer set (Sap2) were dominated by Apha- now quite clear that Peronosporomycetes share no close nomyces species. Neighbor-joining analyses revealed the evolutionary relationships with the true fungi [4, 59, 62, presence of additional taxa that could not be identified 67]. Rather, they are closely related to the heterokont with known Peronosporomycete species represented in algae and hyphochytrids [5, 6, 68]. GenBank. Sequence diversity and mean sequence diver- Peronosporomycetes are currently classified within gence () within bait communities were lower than the the newly erected Kingdom Straminipila (previously diversity within soil communities. Furthermore, the known as Chromista) [8, 18], which is believed to rep- composition of Peronosporomycete communities dif- resent one of the more diverse assemblages of organisms fered among the three fields sampled and between bait on earth [5]. Two distinct subclasses exist within the Pe- and soil communities based on Fst and parsimony tests. ronosporomycetes: the Peronosporomycetidae and the The results of our study represent a significant advance Saprolegniomycetidae. This classification is well sup- in the study of Peronosporomycetes in terrestrial ported by numerous molecular phylogenetic studies habitats. Our work has shown the utility of culture- based on 18S rDNA [22], ITS sequences [12], cyto- independent approaches using 28S rRNA genes to assess chrome oxidase II [11, 31, 43], and, increasingly, 28S the diversity of Peronosporomycete communities in rDNA [48–50]. Although estimates vary, there are now association with plants. It also reveals the presence of around 1200 known species in 88 genera [18]. The rela- potentially new species of Peronosporomycetes in soils tionships among some genera and species are still and plant rhizospheres. uncertain [10, 27, 32, 37, 49, 73]. Despite the diversity and importance of the Perono- Correspondence to: Eric B. Nelson; E-mail: [email protected] sporomycetes, little is known of their distribution and

36 DOI: 10.1007/s00248-005-0187-y & Volume 51, 36–50 (2006) & * Springer Science+Business Media, Inc. 2006 J.M. ARCATE ET AL.: PERONOSPOROMYCETE COMMUNITY DIVERSITY 37 roles in various habitats, and few contemporary ecol- being made from analyses of fungal [53, 71] and other ogical studies of this group have been conducted. This is stramenopile communities [44, 45]. However, primer partly because of the fact that Peronosporomycetes sequences used for analysis of these communities have seldom show up in standard culture-based methods not been effective in detecting Peronosporomycetes [58, commonly used for isolating true fungi from environ- 70], and no molecular-based approach has yet been used mental samples. Instead, a variety of baiting techniques to study a broad range of Peronosporomycetes in [26] have become the standard means of isolating terrestrial samples. Peronosporomycetes from environmental samples and The purpose of this study was to utilize a molecular determining their occurrence, distribution, and diversity. ecological approach to test the hypothesis that direct However, baiting has a number of shortcomings when DNA extractions and amplifications from rhizosphere used to assess species occurrence and distribution. First soil give rise to Peronosporomycete communities that and most importantly are biases because of selective differ from those determined by traditional baiting. We colonization and development on different types of baits tested this hypothesis in soils with different cropping [36, 55]. Even if a particular species can initially colonize histories and planted with different plant species. baits, some species may competitively exclude others during the incubation process, leaving relatively few species to dominate baits. The number of species that Materials and Methods have traditionally been described from baits is relatively limited; generally, fewer than 10 species have been Sampling Site. Samples were collected on 10 Sep- described from a given sample in most studies. Second, tember 2003 from a Howard gravelly loam soil (pH 5.5) the very nature of the baiting system selects species that at the Cornell Vegetable Research Facility, Freeville, NY. produce zoospores under the conditions of the labora- Soils were planted to tomato (Lycopersicon esculentum), tory incubation [14], leaving nonzoospore-producing butternut squash (Cucurbita moschata), or sorghum species or species not developmentally in a state to (Sorghum spp.). Sampled soils were collected from release zoospores to go undetected. Of particular impor- adjacent fields, all of which were in nearly identical mi- tance in the latter case are oospore populations. Because croclimates and with identical soil types. However, each oospores serve as survival structures and likely constitute field had a different cropping history. The tomato field a large proportion of Peronosporomycete biomass in had been in a tomato and winter rye (Secale cereale) soils [16], assessments of diversity based on baiting are rotation for several consecutive years. The winter rye had likely to be underestimated. been plowed under before the tomato crop was planted. Many of the seminal studies of Peronosporomycete The sorghum field was in an alternating year rotation diversity were conducted between the 1920s and the with potatoes (Solanum tuberosum). However, 2003 was 1970s [15]. These studies consisted largely of surveys the first year that sorghum was grown, and for all previous conducted in various terrestrial habitats, ranging from odd years, the crop was rye. The butternut squash field natural forested and grassland sites to swamps, ditches, cropping history was inconsistent. Butternut squash and littoral mud, and agricultural soils (e.g., [1, 19, 25, 34, winter rye were planted in both 2002 and 2003, but the 35]). What emerges from these and other studies is that preceding years had been planted with a range of crops Peronosporomycetes are worldwide in their distribution including melon (Cucumis melo), peppers (Capsicum and are found in nearly all soil types and soil habitats. spp.), sweet corn (Zea mays), tomato, sorghum, and rye. However, the factors that regulate their occurrence and Rhizosphere soil samples from tomato and squash distribution in terrestrial habitats are unknown. It has were collected from five randomly selected plants been suggested that some terrestrial Peronosporomycete established in rows. Soil adjacent to the roots of species might be restricted to particular habitats because individual plants in each of four replicate rows was of the type of vegetation cover [3]. A growing body of removed to a depth of 15 cm. Sorghum rhizosphere soils evidence from other microbes indicates that plants were sampled on a diagonal transect, and four replicate themselves are a major selective force in determining samples were taken from randomly selected plants along the nature of the microbial communities with which they the transect. Replicate samples from each plant species associate (e.g., [41, 56, 57]). Among Peronosporomy- were combined for a total soil volume of approximately cetes, few data are available, but there is good evidence 0.5 L per plant species. These combined soils were each that Pythium species have a profound impact on the thoroughly mixed by repeated turning and shaking in spatial distribution of cherry trees [46, 47]. polyethylene bags. Samples were transported to the Recent molecular ecology studies of bacterial com- laboratory and frozen until use. munities in terrestrial habitats have revealed an incred- ible diversity of previously nondescribed and potentially Baiting. Peronosporomycetes were obtained from nonculturable organisms [61]. Similar discoveries are each of the three rhizosphere soil samples using a mod- 38 J.M. ARCATE ET AL.: PERONOSPOROMYCETE COMMUNITY DIVERSITY ification of traditional baiting techniques [26]. Two Peronosporomycetidae at annealing temperatures below grams of rhizosphere soil were placed into a sterile 60 56-C. 15 mm petri dish and flooded with 20 mL sterile water. Three scored and autoclaved hempseeds were added to DNA Extraction and PCR Conditions. Peron- each dish and were then incubated in darkness at 18-C osporomycete DNA was isolated both from rhizosphere for 48 h. Hempseeds were then transferred aseptically to soil samples and baits using Ultracleani Soil DNA an antibiotic solution [25] and were then incubated at Isolation Kits (MoBio Laboratories, Inc.) according to 18-C in darkness for 14 days. The antibiotic solution the manufacturer’s instructions. For DNA extractions contained per 1 L sterile distilled deionized H2O (ddw) from rhizosphere soil, 0.5 g of soil was used. DNA 0.2 mL pimaricin to suppress fungi and 100 mg am- was also extracted from three hempseed baits for each picillin to suppress bacteria. DNA was then extracted rhizosphere soil sample. DNA was further purified directly from baits. In some preliminary experiments, with the Ultracleani PCR Clean-Up Kit (MoBio Labo- baits were plated on water agar containing 50 mg/mL ratories, Inc.). Two replicate extractions were conducted rifampicin and penicillin G (to suppress bacteria and for subsequent amplification, cloning, and sequencing. fungi, respectively) and were then incubated for 4 days to Polymerase chain reactions (PCRs) for the Oom1 recover viable Peronosporomycete cultures. primer set contained 10 mM Trizma HCl, pH 8.3, 50 mM KCl, 2.5 mM MgCl2, 200 mM of each deoxyribonu- Peronosporomycete Primer Design. A50 region of cleotide triphosphate (dNTP), 0.2 mM each of Oom1f the large subunit rDNA that spans the variable D1 and and Oom1r, 2 U of Taq DNA polymerase, and 1.0 mL D2 regions of the 28S rRNA gene [7] was chosen for template DNA per 25-mL reaction. DNA was amplified amplification using the primer sets Oom1 and Sap2. This with a Bio-Rad MyCycleri thermal cycler using the region was chosen because there are well-developed, following program: initial denaturation at 94-Cfor5 taxonomically defined datasets available for it and min, followed by 35 cycles of denaturation at 94-Cfor30s, because it allows for phylogenetic examination at both annealing at 58-C for 30 s, extension at 72-C for 30 s, and a broad and narrow taxonomic scales. final extension at 72-C for 5 min. PCR products were either The Oom1 primer set was designed from a consensus used immediately or stored at 4-C prior to subsequent sequence derived from a broad range of Peronospor- analyses. Two replicate reactions were run for each sample. omycete species spanning the Peronosporomycetidae and PCRs for the Sap2 primer set contained 10 mM the Saprolegniomycetidae. The Sap2 primer set, on the Trizma HCl, pH 8.3, 50 mM KCl, 2.5 mM MgCl2, other hand, was designed from a consensus sequence 0.2 mM of each of Sap2f and Sap2r, 200 mM of each derived solely from members of the Saprolegniomyceti- dNTP, 4 U of Taq DNA polymerase, and 1.0 mL template dae. Primers were designed using the program Primer DNA per 50-mL reaction. DNA was amplified using the Select 5.07 (DNASTAR, Madison, WI). The Oom1 following PCR program: initial denaturation at 94-C primer set (Oom1F 50-GTGCGAGACCGATAGCGA for 5 min, followed by 35 cycles of denaturation at 94-C ACA-30 and Oom1R 50-TCAAAGTCCCGAACAGCAAC for 30 s, annealing at 56-C for 30 s, extension at 72-C for AA-30) is located between positions homologous to 348 30 s, and a final extension at 72-C for 10 min. and 816 of the Phytophthora megasperma (GenBank Two replicate extractions and amplifications with the X75631) 28S rRNA gene [67] and amplifies a 468-bp Oom1 primer set were conducted to determine whether 0 product. The Sap2 primer set (Sap2F 5 -AGCATAGCGA the sampled communities were the same. Both Fst sta- TTTGGGATAAGTC-30 and Sap2R 50-GTAGGCACCTC tistics and parsimony tests (see below) were not AGTCTCAACCA-30) is located between positions 123 significant (data not shown), indicating that the commu- and 567 of the Saprolegnia ferax (GenBank AF235953) nities arising from the separate extractions were not 28S rRNA gene [48] and amplifies a 444-bp product. significantly different. Sequences from each of these ex- Initial trials of the Oom1 primer set showed a high periments were therefore combined for all subsequent degree of specificity for Peronosporomycetes over other analysis. Amplicons from the Sap2 primer set were only eukaryotes and prokaryotes at annealing temperatures obtained from one DNA extraction. above 56-C. Although this primer pair amplifies 28S rDNA sequences from a broad range of Peronosporo- Cloning and Sequencing of 28S rRNA Genes. mycete genera from both the Peronosporomycetidae and Polymerase chain reaction products from rhizosphere soils Saprolegniomycetidae subclasses when screened in the and baits were cloned using INVaF0 competent cells \ \ laboratory, amplifications from soils and baits were with the pCR 2.1 vector from the TA Cloning Kit somewhat biased toward members of the subclass (Invitrogen, Carlsbad, CA). To concentrate the DNA, two Peronosporomycetidae. Similarly, the Sap2 primer pair 50-mL PCRs for each sample were pooled and were then was biased toward members of the Saprolegniomycetidae concentrated during purification using the MoBio but also amplified 28S rRNA genes from members of the UltraCleani PCR Clean-up Purification Kit (MoBio J.M. ARCATE ET AL.: PERONOSPOROMYCETE COMMUNITY DIVERSITY 39

Laboratories, Inc.). This step insured that the PCR product quent phylogenetic analysis. They were selected to was free of excess salts, dNTPs, and primers, and that we broadly represent the diversity of known Peronospor- could obtain at least 6 ng DNA in 1 mLofproduct.A omycetes and to complement (based on BLAST searches) ligation protocol of 1:1 vector-to-insert ratio yielded the specific groups of our observed sequences. The sequences best cloning efficiency results. Forty-eight clones of each were derived from a variety of Peronosporomycetes, sample were picked into 96-well deep-well plates containing Luria–Bertani and kanamycin and were incubated for 16 hat37-C shaking at 225 rpm. Plasmids were then purified \ using the Wizard SV 96 Plasmid DNA Purification Table 1. Reference taxa used in phylogenetic analyses System (Promega, Madison, WI). Namea GenBank accession no. To determine if the insert was present, plasmid DNA Achlya treleaseana AF119584 0 was amplified using the M13 primer set [M13f (5 - Aphanomyces cochlioides AF218194 GTAAAACGACGGCCAG-30) and M13r (50-CAGGAAA Aphanomyces euteiches AF235939 CAGCTATGAC-30)]. PCRs for the M13 primer set Aphanomyces laevis AF218198 consisted of 10 mM Trizma HCl, pH 8.3, 50 mM KCl, Aphanomyces stellatus AF119587 Aplanes androgynus AF119588 2.5 mM MgCl2, 200 mM of each dNTP, 0.2 mM of each of Aplanopsis spinosa AF119589 M13f and M13r primers, 1 U of Taq DNA polymerase, Apodachlya brachynema AF235936 and 0.5 mL template DNA per 25-mL reaction. DNA was Apodachlya minima AF235937 amplified using the following program: initial denatur- Brevilegnia bispora AF235942 ation at 94-C for 5 min, followed by 35 cycles of Brevilegnia megasperma AF119592 - - Calyptralegnia achlyoides AF119593 denaturation at 94 C for 30 s, annealing at 50 C for 30 s, Dictyuchus monosporus AF119595 extension at 72-C for 30 s, and a final extension at 72-C Dictyuchus sterilis AF218193 for 5 min. PCR products were electrophoresed to screen Hyphochytrium catenoides X80345 for the presence of the insert. Isoachlya toruloides AF235947 Purified plasmid DNA that contained the insert was Leptolegnia caudata AF218176 Leptomitus lacteus AF119597 mixed with the M13f primer (forward direction) and was Pachymetra chaunorhiza AF119598 then submitted to the Cornell Bioresource Center Peronophythora litchii CBS 100.81a Sequencing Facility. Sequencing was performed on an Peronospora parasitica AY035501 b a Applied Biosystems Automated 3730 DNA Analyzer Phytophthora erythroseptica CBS 129.23 using Big Dye Terminator chemistry and AmpliTaq- Phytophthora fragariae AF119601 Phytophthora infestans AF119602 FS DNA Polymerase. Sequences were compiled in Phytophthora megasperma X75631 Sequencher 4.1 (Gene Codes Corp, Ann Arbor, MI, Plectospira myriandra AF119606 USA) or EditSeq 5.07 (DNASTAR), and selected repre- Pythiopsis cymosa AF218172 Pythium aphanidermatum AY598622 sentatives were submitted to GenBank with assigned b accession numbers of AY860252–AY860306. Pythium arrhenomanes AY598628 Pythium capillosum AY598635 Pythium conidiophorum AY598629 Sequence Alignments and Phylogenetic Analyses. Pythium cylindrosporumb AY598643 Raw sequences were edited using EditSeq to manually Pythium graminicolab AY598625 b remove vector sequences and eliminate poorly resolved Pythium insidiosum AY598637 Pythium intermediumb AY598647 regions. These edited sequences were aligned using either b Pythium macrosporum AY598646 MegAlign 5.07 (DNASTAR) or Clustal X version 1.83 Pythium monospermum AY598621 [9], both using the Clustal W algorithms [60]. Separate Pythium multisporumb AY598641 alignments were generated for sequences obtained from Pythium oligandrumb AY598618 DNA extracted from baits, sequences obtained from DNA Pythium paroecandrum AY598644 Pythium salpingophorum AY598630 extracted directly from soil, and combined bait and soil b Pythium sylvaticum AY598645 sequences. A number of tomato rhizosphere sequences Pythium torulosumb AY598624 obtained from DNA extracted directly from soil could Pythium ultimum var. ultimum AY598657 not be used in these analyses because they were too Pythium vanterpoolii AY035534 divergent to be aligned to other soil and bait sequences. Saprolegnia anisopora AF119609 Although BLAST analysis suggested that they were Saprolegnia litoralis AF235952 Sclerospora graminicola AY273987 Peronosporomycete sequences (best BLAST hits were Scoliolegnia asterophora AF119619 AY035537 Pythium sp. AR235 and AF119607 Pythium sp. Thraustotheca clavata AF235951 AR100), they were eliminated from subsequent analyses. aSequences courtesy of Dr. Andre Levesque, Agriculture and Agri-food Ribosomal DNA sequences from reference taxa Canada, Ottawa. (Table 1) were included in alignments used for subse- bType strain. 40 J.M. ARCATE ET AL.: PERONOSPOROMYCETE COMMUNITY DIVERSITY especially Pythium species for which no GenBank se- and plant species or extraction source was then deter- quences were available. These sequences were kindly mined as the number of character changes between the provided by Dr. Andre´ Levesque (Agriculture and Agri- two extraction sources or between two different plant Food Canada, Ottawa) and have recently been described species that could explain the observed distribution of [38]. Additionally, we included selected GenBank Peronosporomycetes. The significance of the observed sequences, which were obtained from our BLAST search covariation was established by determining the expected results. The 28S rDNA sequence from Hyphochytrium number of changes under the null hypothesis that the catenoides (GenBank X80345) was used as an outgroup in communities from which sequences were sampled do not this study [68]. After initial alignments in MegAlign, covary with phylogeny. The null expectation can be sequence alignments were manually edited using BioEdit estimated by assuming that the community identity of 6.0.7 [29] to correct misaligned sequences and ambigu- individual sequences remains fixed, and that the relation- ous base designations. During this final editing, all ships among sequences are randomized [40]. If the sequences were trimmed to a fixed length of 492 bp observed number of transitions from one community (gaps included). In initial alignments, all known 28S to another is less than 95% of the values generated from rDNA sequences from Pythium species were included randomized data, then microbial composition differs [38]. For subsequent phylogenetic analyses, only those significantly between the two communities [42]. The standards with apparent associations with our unknown following direct DNA extraction comparisons were sequences were included. analyzed: tomato vs squash communities, squash vs Phylogenetic analyses were conducted using the sorghum communities, and sorghum vs tomato commu- neighbor-joining (NJ) method [51] as implemented in nities. Additionally, comparisons were made between TreeCon 1.3b [63, 66]. Branch support was based on baited communities and direct DNA extracted commu- 1000 bootstrap replications. Gaps and missing or nities from each of the plant species comparisons and ambiguous data were ignored. Nucleotide substitution from datasets where sequences from all plant species rates for each alignment were calibrated as described by were combined. In all cases, the null expectation was Van de Peer and De Wachter [64, 65] using TreeCon. based on 100 random trees.

Comparative Analysis of Peronosporomycete Com- munities. We used phylogenetic methods described by Results Martin [42] for comparing directly extracted and baited Our analyses using the Oom1 primer set were based on communities in the three different plant rhizospheres. two replicate DNA extractions, whereas analyses from Various measures of diversity were calculated for each the Sap2 primer set were based on only one extraction. Peronosporomycete community. Sequence diversity, nu- We analyzed the two replicate sets of Oom1 sequences cleotide diversity, and  were calculated using the  to determine whether they were sampled different por- program Arlequin 2.001 [54]. For the purpose of these tions of the rhizosphere communities. F analysis of analyses, operational taxonomic units (OTUs) were st the combined soil and bait datasets indicated that no defined as any set of sequences that differed by 1% or differences in sequence composition existed between less. the two replicate extractions and amplifications (F = The degree of differentiation among communities st 0.007, P = 0.13). Therefore sequences from both was estimated by calculating the F statistic as follows: st extractions were combined for all subsequent analyses. Fst =(T – W)/T, where T is the genetic diversity for all of the communities combined (e.g., all combined plant Phylogenetic Analyses of Oom1-derived Soil Com- species communities or all combined soil and bait com- munities. Our phylogenetic analyses of Oom1-ampli- munities) and  is the genetic diversity within each of W fied soil communities revealed the presence of a number the individual communities averaged over all of the com- of well-supported clusters, many of which were closely munities [42]. To calculate this statistic, aligned sequences related to 28S rDNA sequences of known Peronospo- without reference taxa were directly imported into romycete species. Sequences grouped largely into two Arlequin 2.001. The statistical significance of F was st main clusters of Pythium species (Pythium cluster I and estimated from 1000 permutations at a significance level Pythium cluster II; Fig. 1). However, some sequences, of 0.05. exclusively from tomato rhizospheres, grouped with a The parsimony test, for which the theory has been Phytophthora/Peronospora cluster, whereas only one se- described by Maddison and Slatkin [40], was carried out quence from squash grouped with the Saprolegniomyce- by generating sets of 100 phylogenetic trees on which tidae (see arrow). plant species or extraction source was optimized as a discrete character using parsimony with the aid of Pythium Cluster I. A number of taxa largely from Mesquite 1.02 [39]. The covariation between phylogeny tomato rhizospheres clustered with Pythium torulosum J.M. ARCATE ET AL.: PERONOSPOROMYCETE COMMUNITY DIVERSITY 41 42 J.M. ARCATE ET AL.: PERONOSPOROMYCETE COMMUNITY DIVERSITY

(Fig. 1; Pythium cluster IA). Another major group of Pythium Cluster I. This cluster corresponds to the same sequences exclusively from sorghum rhizospheres Pythium cluster I found among the soil communities. As represented closely related strains of P. arrhenomanes or with soil communities, bait-derived sequences in this P. aristosporum (only P. arrhenomanes shownintree;Fig.1; cluster were dominated by a large group of nearly iden- Pythium cluster IC). Other groups of sequences in Pythium tical sequences that came from the rhizospheres of all cluster I could not be grouped with any 28S rDNA se- three plant species and were very closely related to P. quence from known Pythium species. Sequences in Pythium torulosum (Fig. 2; Pythium cluster IA). Another group of cluster IB were exclusively from squash rhizospheres, sequences exclusively from tomato rhizospheres whereas those from Pythium clusters ID and IE were from (Pythium cluster IC) was nearly identical to P. monosper- mixed plant rhizospheres and sorghum rhizospheres, re- mum, whereas most other sequences within this cluster spectively. Despite the close affinities of taxa in Pythium (e.g., Pythium cluster IB and other individual sequences) cluster ID to P. aphanidermatum and P. monospermum and could not be assigned to any known taxa. taxa in Pythium cluster IE to P. insidiosum, they all likely Phytophthora/Peronospora and Saprolegniomycetidae represent distinct species of Pythium that are either novel or Clusters. One tomato sequence grouped in the Phy- are known species for which no 28S rDNA sequences are tophthora/Peronospora cluster and nearly identical to available. Phytophthora infestans (Fig. 2). No Peronospora or Sa- Phytophthora/Peronospora Cluster. Some sequences prolegniomycetidae sequences were found on baits from from tomato rhizospheres grouped with species of Phy- rhizospheres. tophthora and Peronospora (Fig. 1). One sequence was Pythium Cluster II. Sequences from this cluster fell essentially identical to Phytophthora infestans, whereas four largely into two groups, neither of which was closely other tomato sequences were similar to that of Pero- affiliated with any known Pythium species. Pythium clus- nospora parasitica and likely represent other Peronospora ter IID represented a group of taxa from tomato rhizo- species. spheres (Fig. 2), whereas those in Pythium cluster E Pythium Cluster II. All sequences in this cluster re- consisted exclusively of sequences from sorghum rhizo- presented unknown Pythium species (Fig. 1; Pythium spheres with close relationships to P. intermedium, P. cluster IF). Although they grouped loosely with P. inter- paroecandrum, P. cylindrosporum, and P. sylvaticum. medium, P. macrosporum, P. sylvaticum, P. paroecan- drum,andP. cylindrosporum,theylikelyrepresent Phylogenetic Analyses of Sap2-Amplified Communities. distinct species of Pythium that are either novel or are Analysis of Sap2-derived communities revealed a general known species for which no 28S rDNA sequences are lack of sequence diversity among all rhizosphere samples. available. The majority of sequences fell into two major clusters, Saprolegniomycetidae. Only one sequence from regardless of whether they originated from baits or directly squash rhizospheres clustered with taxa in the Sapro- from soils (Fig. 3). Many of the squash and sorghum se- legniomycetidae (Fig. 1; see arrow). This sequence was quences amplified from baits or directly from soils clus- most closely related to Apodachlya brachynema but likely tered with species of Aphanomyces, Plectospira,and represents a distinct genus within this group. Pachymetra (Aphanomyces cluster I), with the greatest similarity (and BLAST hits) to Aphanomyces cochlioides. Phylogenetic Analyses of Oom1-Amplified Bait However, some squash and all tomato bait sequences Communities. Similar to soil communities, neighbor- clustered with members of the Peronosporomycetidae joining trees of Oom1-amplified bait communities also most closely aligned with Pythium ultimum var. ultimum revealed a number of unique clusters, many of which (Fig. 3; Pythium cluster III). were closely related to 28S rDNA sequences of known Peronosporomycete species (Fig. 2). As with soil com- Comparison of Bait and Soil Peronosporomycete munities, sequences from baits fell into the same two Community Diversity. In all community comparisons, major Pythium clusters (clusters I and II). the number of OTUs shared between the two com-

Figure 1. Neighbor-joining analysis of 86 partial 28S rDNA sequences obtained from Oom1 amplifications of DNA extracted directly from rhizosphere soils (soil communities). Ribosomal DNA sequences from reference taxa were included to anchor sequences of unknown affiliation, where possible, to known Peronosporomycete species. Genetic distances were calculated using calibrated substitution rates (0.34) as described previously [64, 65]. Trees were rooted with the hyphochytrid Hyphochytrium catenoides (X80345). Sequence designations beginning with 1 are from the first extraction; those beginning with 2 are from the second extraction. Numbers at each node represent bootstrap values based on 1000 replications. J.M. ARCATE ET AL.: PERONOSPOROMYCETE COMMUNITY DIVERSITY 43 44 J.M. ARCATE ET AL.: PERONOSPOROMYCETE COMMUNITY DIVERSITY munities was low, generally less than 5–10%. Sequence Sequence diversity was similar among all pairwise com- diversity was high and significantly greater in soil com- parisons using the Oom1 primer set. However, the Sap2 munities than in bait communities. For example, sig- primer set yielded lower levels of sequence diversity. nificantly greater sequence diversity was observed in soil No differences in nucleotide diversity were observed communities of all three combined plant species and among soil communities using the Oom1 primers, re- from sorghum and squash soil communities than from gardless of the plant species. However, among bait com- the respective bait communities (Table 2). This was true munities, nucleotide diversity was greatest in tomato for both primer sets. rhizospheres, followed by sorghum then squash rhizo- Nucleotide diversity was low among all Oom1- spheres. With Sap2 primers, greater nucleotide diversity derived communities but much higher among Sap2- was observed in bait communities from tomato rhizo- derived communities. Significantly greater nucleotide spheres than in those from sorghum or squash rhizo- diversity was observed among Oom1 sequences from spheres. Much higher levels of nucleotide diversity were squash soil communities than from squash bait commu- found among soil communities than among bait com- nities. However, these differences were not observed munities using the Sap2 primers. However, no differ- using the Sap2 primer set. With the Sap2 primer set, ences were observed among the three plant species. significantly greater nucleotide diversity was found in the Overall sequence divergence  was generally greater sorghum and tomato soil communities than in the from soil communities than from bait communities respective bait communities. (Table 3). Differential influences of the plant/cropping The mean sequence divergence,  (a measure of system on Peronosporomycete communities were ob- community diversity), was generally lower among Oom1- served for both bait and soil communities, regardless of derived communities than with Sap2-derived communities. the primer set used. For example, Oom1-derived bait Diversity of Oom1-derived bait and soil communities from communities from tomato and sorghum rhizospheres sorghum and tomato rhizospheres did not differ. However, were significantly more diverse than those from squash significantly greater diversity was found in squash soil rhizospheres. However, with the Sap2 primer set, bait communities than in squash bait communities. Similarly, communities from tomato rhizospheres were more di- significantly greater diversity was found in Sap2-derived verse than those from either sorghum or squash. Among tomato and sorghum soil communities than in the respec- soil communities, no differences in overall sequence di- tive bait communities. vergence were apparent among the different plants/crop- Fst statistics for pairwise comparisons of bait and soil ping systems, regardless of the primer set used. However, communities in all plant rhizospheres were highly Sap2-derived soil communities from tomato rhizo- significant (P G 0.001), regardless of the primer set used. spheres were significantly more diverse than those from Furthermore, the Fst statistics of combined sequences squash but not sorghum rhizospheres. from rhizospheres of all plant species were also signifi- Fst statistics varied considerably depending on the cant (P G 0.001), indicating that bait communities were specific plant comparison and on the bait vs soil commu- significantly different from soil communities. nity comparison. Fst values calculated from comparisons of Parsimony tests of differentiation between soil and tomato and sorghum communities showed significant bait communities were all highly significant (P G 0.001), community divergence when determined with Oom1- regardless of the plant species or primer set tested. Each derived soil communities and Sap2-derived bait and soil of the pairwise comparisons among plant species was communities but not with Oom1-derived bait communi- also highly significant (P G 0.03; data not shown). ties. Fst values calculated from comparisons of tomato and squash communities showed significant community di- Comparison of Peronosporomycete Community Di- vergence when determined with Oom1-derived soil and versity in the Rhizosphere of Different Plant Species bait communities and Sap2-derived soil communities but Analyses of all pairwise comparisons among the three not with Sap2-derived bait communities. Fst values plant species are shown in Table 3. As with the soil vs calculated from comparisons of sorghum and squash bait comparisons, the number of OTUs shared between rhizosphere communities indicated significant community the rhizosphere communities of each of the paired plant divergence only with Oom1- and Sap2-derived bait species was low and was in the range of less than 5–10%. communities and not with soil communities.

Figure 2. Neighbor-joining analysis of 89 partial 28S rDNA sequences obtained from Oom1 amplifications of DNA extracted from hempseed baits incubated in rhizosphere soils (bait communities). Ribosomal DNA sequences from reference taxa were included to anchor sequences of unknown affiliation, where possible, to known Peronosporomycete species. Genetic distances were calculated using calibrated substitution rates (0.36) as described previously [64, 65]. Trees were rooted with the hyphochytrid H. catenoides (X80345). Sequence designations beginning with 1 are from the first extraction; those beginning with 2 are from the second extraction. J.M. ARCATE ET AL.: PERONOSPOROMYCETE COMMUNITY DIVERSITY 45 46 J.M. ARCATE ET AL.: PERONOSPOROMYCETE COMMUNITY DIVERSITY

Table 2. Comparative diversity estimates of directly-extracted and baited Peronosporomycete communities from the rhizospheres of different plant species Sequence Nucleotide Mean sequence Primer Community pairwise Usable Shared diversity diversity divergence g a b c d T e T f T h set comparison OTUs loci OTUs ( SD) ( SD)  ( SD) Fst Oom1 Combined baits (91) 116 481 5 0.956 (0.013) 0.127 (0.061) 61.23 (29.54) 0.114 (P G 0.001) Combined soils (86) 476 0.985 (0.006) 0.127 (0.061) 60.43 (29.18) Oom1 Sorghum baits (33) 42 473 2 0.892 (0.043) 0.122 (0.060) 57.79 (28.65) 0.228 (P G 0.001) Sorghum soils (28) 467 0.981 (0.015) 0.115 (0.057) 53.56 (26.42) Oom1 Tomato baits (32) 43 470 2 0.978 (0.015) 0.109 (0.054) 51.18 (25.29) 0.146 (P G 0.001) Tomato soils (27) 473 0.937 (0.033) 0.131 (0.065) 62.16 (30.83) Oom1 Squash baits (31) 35 450 1 0.837 (0.051) 0.013 (0.007) 5.92 (3.23) 0.352 (P G 0.001) Squash soils (26) 464 0.969 (0.027) 0.123 (0.061) 57.17 (28.43) Sap2 Combined baits (55) 78 515 2 0.945 (0.019) 0.346 (0.167) 178.15 (77.45) 0.125 (P G 0.001) Combined soils (55) 512 0.985 (0.009) 0.489 (0.236) 250.38 (120.61) Sap2 Sorghum baits (17) 27 512 2 0.882 (0.072) 0.090 (0.046) 45.19 (23.08) 0.130 (P G 0.001) Sorghum soils (19) 512 0.983 (0.022) 0.429 (0.215) 219.46 (109.85) Sap2 Tomato baits (20) 28 513 0 0.953 (0.035) 0.289 (0.144) 148.25 (74.10) 0.355 (P G 0.001) Tomato soils (14) 510 0.989 (0.031) 0.615 (0.315) 313.89 (160.45) Sap2 Squash baits (19) 28 515 0 0.860 (0.071) 0.350 (0.175) 180.27 (90.30) 0.279 (P G 0.001) Squash soils (20) 501 0.968 (0.033) 0.332 (0.166) 166.29 (83.08) a Numbers in parentheses represent the total number of sequences analyzed. b OTU = operational taxonomic unit defined as sequences with Q99% nucleotide similarity. c All Oom1 sequences were truncated to 492 nucleotides (gaps included); Sap2 sequences were trimmed to 517 nucleotides (gaps included); number of nucleotides used in the analyses to calculate the various statistics. d OTUs shared between the two samples. e The probability that two randomly chosen sequences are different. f The probability that two randomly chosen homologous nucleotides are different; a measure of diversity within the usable loci. g Total genetic variation in the sample. h Fst =(T – W)/T, where T is the genetic diversity of the combined community samples and W is the genetic diversity within each community averaged over the two communities being compared. Fst is a measure of the level of differentiation between each community and the total combined community.

Discussion extracted directly from rhizosphere soil. This conclusion is based on several lines of evidence. First, our phyloge- The major hypothesis underlying our work was that netic analysis of Oom1-derived communities revealed the terrestrial Peronosporomycete communities described presence of several clusters found among soil communi- using a molecular phylogenetic approach based on direct ties that were absent among bait communities. For DNA extraction and amplification of 28S rRNA genes example, a cluster of sorghum rhizosphere sequences would be different and more diverse than those using nearly identical to Pythium arrhenomanes was found conventional baiting strategies. We reasoned that, in among soil communities (Fig. 2, cluster IC) but not in addition to the vegetative life stages [zoospores, sporan- bait communities from the same rhizosphere soil (see gia, hyphal swellings (or gemmae)] that are typically also Fig. 2, clusters IB and II). Second, sequence diversity present and able to colonize baits, we should also detect was much greater in soil communities than in bait populations of Peronosporomycetes that may exist only communities in all rhizospheres except tomato, regard- as oospores or other populations that might not be less of the primer set used. This was also reflected in the isolated using baiting procedures. In our work, 28S Fst statistics, which indicated that soil communities, rRNA genes were amplified directly from baits as well as regardless of the plant species, were significantly different from soil to avoid problems associated with the mor- from the respective bait communities. Furthermore, phological identification of isolates from baits and also to relatively few OTUs were present in both bait and soil facilitate comparative analyses. communities, indicating that each method samples the Although our sample sizes were small (about 15–25 underlying rhizosphere community differently. sequences per sample), our data clearly show that com- Although the source of the increased diversity in soil munities obtained from hempseed baits exhibit a lower communities is unknown, we hypothesize that it might level of diversity than those obtained from DNA be because of oospore populations of Peronosporomy-

Figure 3. Neighbor-joining analysis of 65 partial 28S rDNA sequences obtained from Sap2 amplifications of DNA extracted directly from rhizosphere soils and from hempseed baits incubated in rhizosphere soils. Ribosomal DNA sequences from reference taxa were included to anchor sequences of unknown affiliation, where possible, to known Peronosporomycete species. Genetic distances were calculated using calibrated substitution rates (0.28) as described previously [64, 65]. Trees were rooted with the hyphochytrid H. catenoides (X80345). J.M. ARCATE ET AL.: PERONOSPOROMYCETE COMMUNITY DIVERSITY 47

Table 3. Diversity estimates of Peronosporomycete communities from the rhizospheres of different plant species based on all pairwise comparisons of each plant species Community Sequence Mean sequence pairwise Usable Shared diversity Nucleotide divergence g a b c d T e T f T h Primer set comparison OTUs loci OTUs ( SD) diversity ( SD)  ( SD) Fst Bait communities Oom1 Tomato (33) 58 469 1 1.000 (0.008) 0.112 (0.055) 52.62 (25.97) 0.457 (P = 0.068) Sorghum (25) 465 1.000 (0.011) 0.054 (0.027) 25.15 (12.73) Oom1 Squash (31) 64 445 2 1.000 (0.008) 0.013 (0.007) 5.60 (3.08) 0.290 (P G 0.001) Tomato (33) 463 1.000 (0.008) 0.115 (0.057) 53.56 (26.42) Oom1 Sorghum (25) 56 457 1 1.000 (0.011) 0.052 (0.022) 23.91 (12.12) 0.830 (P = 0.058) Squash (31) 457 1.000 (0.008) 0.013 (0.007) 5.60 (3.08) Sap2 Tomato (14) 26 526 0 1.000 (0.034) 0.020 (0.011) 10.48 (5.79) 0.959 (P G 0.001) Sorghum (19) 443 1.000 (0.022) 0.003 (0.002) 1.48 (1.06) Sap2 Squash (9) 21 510 1 1.000 (0.052) 0.009 (0.006) 4.61 (2.83) –0.034 (P = 0.911) Tomato (12) 526 1.000 (0.034) 0.020 (0.011) 10.48 (5.79) Sap2 Squash (9) 25 510 0 1.000 (0.052) 0.009 (0.006) 4.61 (2.83) 0.979 (P G 0.001) Sorghum (16) 443 1.000 (0.022) 0.003 (0.002) 1.48 (1.06) Soil communities Oom1 Tomato (27) 60 476 0 1.000 (0.008) 0.129 (0.064) 61.29 (30.41) 0.093 (P = 0.014) Sorghum (33) 476 1.000 (0.008) 0.112 (0.055) 52.51 (25.91) Oom1 Squash (26) 53 473 1 1.000 (0.008) 0.127 (0.063) 59.91 (29.78) 0.057 (P = 0.052) Tomato (27) 474 1.000 (0.008) 0.131 (0.065) 62.32 (30.91) Oom1 Sorghum (33) 59 468 2 1.000 (0.008) 0.112 (0.055) 52.59 (25.95) 0.002 (P = 0.358) Squash (26) 470 1.000 (0.008) 0.123 (0.060) 57.16 (28.43) Sap2 Tomato (14) 29 510 1 0.989 (0.031) 0.615 (0.315) 313.88 (160.45) 0.188 (P G 0.002) Sorghum (19) 512 0.983 (0.022) 0.429 (0.215) 219.46 (109.85) Sap2 Squash (22) 31 501 0 0.970 (0.028) 0.366 (0.182) 183.49 (91.16) 0.208 (P G 0.006) Tomato (14) 510 0.989 (0.031) 0.615 (0.315) 313.88 (160.45) Sap2 Sorghum (19) 40 512 2 0.983 (0.022) 0.429 (0.215) 219.46 (109.85 –0.029 (P = 0.634) Squash (22) 501 0.970 (0.028) 0.366 (0.182) 183.49 (91.16) a Numbers in parentheses represent the total number of sequences analyzed. b OTU = operational taxonomic unit defined as sequences with Q99% nucleotide similarity. c All sequences were truncated to 492 nucleotides (gaps included); number of nucleotides used in the analyses to calculate the various statistics. d OTUs shared between the two samples. e The probability that two randomly chosen sequences are different. f The probability that two randomly chosen homologous nucleotides are different; a measure of diversity within the usable loci. g Total genetic variation in the sample. h Fst =(T – W)/T, where T is the genetic diversity of the combined community samples and W is the genetic diversity within each community averaged over the two communities being compared. Fst is a measure of the level of differentiation between each community and the total combined community. cetes that have gone undetected using baiting procedures. known for this group of organisms. For example, we Based on our preliminary laboratory studies (unpub- observed several unique clusters of Pythium-like sequen- lished), we have shown that our methods can effectively ces that could not be associated with any known Pythium lyse oospores of a variety of Peronosporomycete species, species for which 28S rDNA sequences are available. extract DNA, and amplify our target sequences. We These unique groups were found not only from DNA therefore expected that we were effective in extracting extracted directly from soil but also from DNA extracted oospores present in our test soils. However, it is possible from baits. Traditionally, species have been identified that other Peronosporomycete populations may be less from baits based on the reproductive structures formed responsive to baits or may be less competitive under the under the conditions of the incubation or after isolation bait incubation conditions. Clearly, the choice of baiting in culture [26]. It is likely, therefore, that other species material can influence the types of Peronosporomycete are present on colonized baits, which are not detected species that can be detected [14, 52]. Therefore, it is because they lack diagnostic structures. Furthermore, possible that the choice of different baits would yield given that some clusters were found among bait com- different results of Peronosporomycete diversity. munities that were not detected in soil communities (e.g., Results of this study demonstrate the feasibility of Fig. 3, cluster II sequences nearly identical to Pythium using molecular approaches to study Peronosporomycete ultimum var. ultimum), the use of DNA extractions both communities and to uncover hidden levels of biodiver- from baits and from soils will likely provide a more sity. Our data suggest that molecular characterization is complete picture of the diversity of Peronosporomycete detecting a greater level of diversity than previously communities in soil and aquatic habitats. 48 J.M. ARCATE ET AL.: PERONOSPOROMYCETE COMMUNITY DIVERSITY

A prominent observation from our data was the obtained from rhizospheres of the three plant species significant effect of the plant/cropping system on rhizo- were closely related to P. ultimum, P. torulosum, P. ar- sphere Peronosporomycete communities. For example, a rhenomanes/P. aristosporum, P. monospermum, P. inter- number of individual clusters within the NJ trees of medium, P. sylvaticum, P. paroecandrum,andP. either bait or soil communities were composed of se- cylindrosporum. Those sequences related to P. arrheno- quences from only one plant/cropping system. This is manes/P. aristosporum all came from sorghum rhizo- particularly true for groups of sequences from tomato spheres. This would be predicted because these species and sorghum rhizospheres. The significant impact of are common pathogens of grasses and cereals [69]. plant/cropping system in influencing the Peronosporo- Similarly, sequences closely related to P. torulosum came mycete community is further indicated by the parsimony largely from the rhizosphere of tomato, which is a com- test, which points to a significant (P G 0.001) covariation mon host for P. torulosum [69]. of Peronosporomycete lineage with plant species. Sec- Strikingly absent from our rhizosphere Peronospor- ond, significant Fst statistics for comparisons between omycete communities was a greater diversity of sequenc- squash or sorghum and tomato indicate that these com- es from the Saprolegniomycetidae. Although sequences munities include significantly different sets of taxa. related to Aphanomyces species were detected largely Although there are subtle differences in nucleotide di- from sorghum and squash rhizospheres, and one se- versity and overall sequence divergence between squash quence related to Apodachlya was detected, we anticipat- and sorghum communities, we are not confident in ed a higher frequency of members of this group because concluding that these communities differ. they have been described in association with seeds [74, There are at least two explanations for these ob- 75], and species of related genera are known plant servations: either plants actively select for specific Pero- pathogens [20, 23, 28, 33, 72, 76]. The absence of these nosporomycete communities in their rhizospheres, or, species in our current study may indicate either that (1) because of the different cropping histories, each of the they indeed are not present in the rhizospheres of these three fields sampled contained significantly different plant species, (2) their absence is related to either low Peronosporomycete communities. Although each of the seasonal abundance discussed above or defined micro- three plant species sampled was grown adjacent to each habitats that were not sampled in the course of this study, other in soils with identical soil properties and presum- or (3) our primers were not effective in detecting these ably homogeneous Peronosporomycete communities, the species, despite the ability of these primer sets to detect different cropping histories unique to each field prevent these species in laboratory controls. However, current us from being able to reject the possibility that Perono- studies with Agrostis stolonifera Peronosporomycete com- sporomycete communities differed in these soils, making munities using these same two primer sets indicate an it unclear whether plants per se select for specific Pero- abundance of taxa from the Saprolegniomycetidae nosporomycete communities. Experiments are underway (Nelson, unpublished data). This suggests that our pri- to test this hypothesis more directly. mer sets adequately detect these species if they are present. Despite our abilities to clearly discern differences The results of our study have shown the utility of between bait and soil communities and between rhizo- culture-independent approaches using 28S rRNA genes sphere communities associated with different plant/ to assess diversity of Peronosporomycete communities in cropping systems, our results are still likely to underesti- association with plants. This approach has revealed the mate the diversity of Peronosporomycete communities in presence of as yet unexplored diversity of Peronospor- the rhizosphere. Our sampling intensity was considerably omycetes in plant rhizospheres. Further work in this area limited, with relatively few clones sequenced per sample. will provide a better understanding of the distribution A greater sampling of our clone libraries will undoubtedly and functional roles of Peronosporomycetes in the en- reveal additional community diversity beyond what we vironment as well as their pathogenic interactions in soils detected in our current study. Peronosporomycetes show and water bodies. distinct seasonal fluctuations in species abundance [1, 19, 21, 34], and some species may be highly aggregated [13, 14]. These observations indicate the need for more in- Acknowledgments tensive sampling in time and space to capture the full range of diversity in a particular habitat. Special thanks are due to Dr. Andre´ Levesque for pro- One of the striking characteristics of rhizosphere viding 28S rDNA sequences of a wide range of Pero- Peronosporomycete communities revealed in this study nosporomycete species, especially those of Pythium spp., was the dominance by Pythium species. This is not many of which represented the type or neotype strain. surprising because nearly all surveys of Peronosporomy- We also thank Kathie Hodge for her helpful discussions cetes have revealed an abundance of Pythium species over and review of the manuscript and Michael Milgroom, a wide range of habitats [2, 24, 25, 30, 34, 35]. Sequences Fernando Ponce, Angelika Rumberger, and Sofia Wind- J.M. ARCATE ET AL.: PERONOSPOROMYCETE COMMUNITY DIVERSITY 49 stam for their helpful comments on the manuscript. The 19. Dick, MW, Ali-Shtayeh, MS (1986) Distribution and frequency of work was supported by grants to JMA from the CALS Pythium species in parkland and farmland soils. Trans Br Mycol Soc 86: 49–62 Charitable Trust, The Fredric N. Gabler Memorial 20. Dick, MW, Croft, BJ, Magarey, RC, de Cock, AWAM, Clark, G Research Honors Endowment, and Hatch/Multistate (1989) A new genus of the Verrucariaceae (Oomycetes). Bot J Linn Funds for Undergraduate Student Research. Soc 99: 97–113 21. Dick, MW, Newby, HV (1961) The occurrence and distribution of Saprolegniaceae in certain soils of south-east England. I. Occur- rence. J Ecol 49: 403–419 References 22. Dick, MW, Vick, MC, Gibbings, JG, Hedderson, TA, Lastra, CCL (1999) 18S rDNA for species of Leptolegnia and other Pero- 1. Ali-Shtayeh, MS, Lim Ho, CL, Dick, MW (1986) The phenology of nosporomycetes: justification for the subclass taxa Saproleg- Pythium in soil. J Ecol 74: 823–840 niomycetidae and Peronosporomycetidae and division of the 2. Ali, EH, Abdel-Raheem, A (2003) Distribution of zoosporic fungi Saprolegniaceae sensu lato into the Leptolegniaceae and Saproleg- in the mud of major Egyptian lakes. J Basic Microbiol 43: 175– niaceae. Mycol Res 103: 1119–1125 184 23. Drechsler, C (1927) Two water molds causing tomato rootlet 3. Apinis, AE (1964) Concerning the occurrence of phycomycetes in injury. J Agric Res 34: 287–296 alluvial soils of certain pastures, marshes, and swamps. Nova 24. El-Hissy, F, Khallil, AR, Ali, E (1994) Aquatic phycomycetes from Hedwig 8: 103–126 Egyptian soil (Delta region). Microbiol Res 149: 271–282 4. Baldauf, SL (2003) The deep roots of eukaryotes. Science 300: 25. El-Hissy, FT, Oberwinkler, F (1999) Oomycetes and chytridiomy- 1703–1706 cetes (Mastigomycotina) from different soil habitats in Tu¨bingen 5. Ben Ali, A, De Baere, R, De Wachter, R, Van de Peer, Y (2002) region (Germany). Acta Hydrobiol 41: 139–153 Evolutionary relationships among heterokont algae (the autotro- 26. Fuller MS, Jaworski A (Eds.) (1987) Zoosporic Fungi in Teaching phic stramenopiles) based on combined analyses of small and large and Research, Vol. Southeastern Publishing, Athens subunit ribosomal RNA. Protist 153: 123–132 27. Go¨ker, M, Voglmayr, H, Riethmuller, A, Weiss, M, Oberwinkler, F 6. Ben Ali, A, De Baere, R, Van der Auwera, G, De Wachter, R, Van (2003) Taxonomic aspects of Peronosporaceae inferred from de Peer, Y (2001) Phylogenetic relationships among algae based on Bayesian molecular phylogenetics. Can J Bot 81: 672–683 complete large-subunit rRNA sequences. Int J Syst Evol Microbiol 28. Haber, S, Barr, DJS, Platford, RG (1991) Observations on the 51: 737–749 distribution of flame chlorosis and its association with certain 7. Ben Ali, A, Wuyts, J, De Wachter, R, Meyer, A, Van de Peer, Y zoosporic fungi and the intensive cultivation of cereals. Can J Plant (1999) Construction of a variability map for eukaryotic large Pathol 13: 241–246 subunit ribosomal RNA. Nucleic Acids Res 27: 2825–2831 29. Hall, TA (1999) BioEdit: A user-friendly biological sequence 8. Cavalier-Smith, T (1986) The kingdom Chromista, origin and alignment editor and analysis program for Windows 95/98/NT. systematics. In: Round, FE, Chapman, DJ (Eds.) Progress in Nucleic Acids Symp Ser 41: 95–98 Phycological Research, Vol 4. Biopress, Bristol, pp 309–347 30. Hassan, SKM, Fadl-Allah, EM (1992) Studies on some zoosporic 9. Chenna, R, Sugawara, H, Koike, T, Lopez, R, Gibson, TJ, fungi in soils of upper Egypt. Acta Mycol 27: 159–170 Higgins, DG, Thompson, JD (2003) Multiple sequence alignment 31. Hudspeth, DSS, Nadler, SA, Hudspeth, MES (2000) A COX2 with the Clustal series of programs. Nucleic Acids Res 31: 3497– molecular phylogeny of the peronosporomycetes. Mycologia 92: 3500 674–684 10. Constantinescu, O, Fatehi, J (2002) Peronospora-like fungi 32. Hudspeth, DSS, Stenger, D, Hudspeth, MES (2003) A cox2 (Chromista, Peronosporales) parasitic on Brassicaceae and related phylogenetic hypothesis for the downy mildews and white rusts. hosts. Nova Hedwig 74: 291–338 Fungal Divers 13: 47–57 11. Cook, KL, Hudspeth, DSS, Hudspeth, MES (2001) A cox2 33. Jee, H-J, Ho, H-H, Cho, W-D (2000) Pythiogeton zeae sp. nov. phylogeny of representative marine peronosporomycetes (Oomy- causing root and basal stalk rot of corn in Korea. Mycologia 92: cetes). Nova Hedwig 122: 231–243 522–527 12. Cooke, DEL, Drenth, A, Duncan, JM, Wagels, G, Brasier, CM 34. Khallil, AM, Elhissy, FT, Ali, EH (1995) Seasonal fluctuations of (2000) A molecular phylogeny of Phytophthora and related aquatic fungi recovered from Egyptian soil (Delta region). J Basic Oomycetes. Fungal Genet Biol 30: 17–32 Microbiol 35: 93–102 13. Dick, MW (1962) The occurrence and distribution of Saproleg- 35. Khulbe, RD (1991) An ecological study of watermolds of forest niaceae in certain soils of south-east England. II. Distribution soils of Kumaun Himalaya India. Trop Ecol 32: 127–135 within defined areas. J Ecol 50: 119–127 36. Klich, MA, Tiffany, LH (1985) Distribution and seasonal occur- 14. Dick, MW (1966) The Saprolegniaceae of the environs of Blelham rence of aquatic Saprolegniaceae in northwest Iowa. Mycologia 77: Tarn: Sampling techniques and the estimation of propagule 373–380 numbers. J Gen Microbiol 42: 257–282 37.Leclerc,MC,Guillot,J,Deville,M(2000)Taxonomicand 15. Dick, MW (1976) The ecology of aquatic phycomycetes. In: Gareth phylogenetic analysis of Saprolegniaceae (Oomycetes) inferred Jones, EB (Ed.) Recent Advances in Aquatic Mycology. John Wiley from LSU rDNA and ITS sequence comparisons. Antonie Van and Sons, New York, pp 513–542 Leeuwenhoek 77: 369–377 16. Dick, MW (1992) Patterns of phenology in populations of 38. Levesque, CA, de Cock, AWAM (2004) Molecular phylogeny and zoosporic fungi. In: Carroll, GC, Wicklow, DT (Eds.) The Fungal taxonomy of the genus Pythium. Mycol Res 108: 1363–1383 Community. Its Organization and Role in the Ecosystem. Marcel 39. Maddison WP , Maddison DR (2004) Mesquite: A modular system Dekker, Inc., New York, pp 355–382 for evolutionary analysis, http://mesquiteproject.org/mesquite/ 17. Dick, MW (2001) The Peronosporomycetes. In: McLaughlin, DJ, mesquite.html. McLaughlin, EG, Lemke, PA (Eds.) Systematics and Evolution, 40. Maddison, WP, Slatkin, M (1991) Null models for the number of Part A, Vol VII. Springer-Verlag, Berlin, pp 39–72 evolutionary steps in a character on a phylogenetic tree. Evolution 18. Dick, MW (2001) Straminipilous Fungi, Vol. Kluwer Academic 45: 1184–1197 Publishers, Dordrecht 41. Marschner, P, Yang, CH, Lieberei, R, Crowley, DE (2001) Soil and 50 J.M. ARCATE ET AL.: PERONOSPOROMYCETE COMMUNITY DIVERSITY

plant specific effects on bacterial community composition in the ature gradient gel electrophoresis. Appl Environ Microbiol 65: rhizosphere. Soil Biol Biochem 33: 1437–1445 2614–2621 42. Martin, AP (2002) Phylogenetic approaches for describing and 59. Stechmann, A, Cavalier-Smith, T (2003) Phylogenetic analysis of comparing the diversity of microbial communities. Appl Environ eukaryotes using heat-shock protein Hsp90. J Mol Evol 57: 408– Microbiol 68: 3673–3682 419 43. Martin, FN (2000) Phylogenetic relationships among some 60. Thompson, JD, Higgins, DG, Gibson, TJ (1994) Clustal W: Pythium species inferred from sequence analysis of the mito- Improving the sensitivity of progressive multiple sequence align- chondrially encoded cytochrome oxidase II gene. Mycologia 92: ment through sequence weighting, position specific gap weighting, 711–727 and weight matrix choice. Nucleic Acids Res 22: 4673–4680 44. Massana, R, Castresana, J, Balague, V, Guillou, L, Romari, K, 61. Torsvik, V, Ovreas, L, Thingstad, TF (2002) Prokaryotic diversi- Groisillier, A, Valentin, K, Pedros-Alio, C (2004) Phylogenetic and ty—Magnitude, dynamics, and controlling factors. Science 296: ecological analysis of novel marine stramenopiles. Appl Environ 1064–1066 Microbiol 70: 3528–3534 62. Van de Peer, Y, Baldauf, SL, Doolittle, WF, Meyer, A (2000) An 45. Moreira, D, Lopez-Garcia, P (2002) The molecular ecology of updated and comprehensive rRNA phylogeny of (crown) eukar- microbial eukaryotes unveils a hidden world. Trends Microbiol yotes based on rate-calibrated evolutionary distances. J Mol Evol 10: 31–38 51: 565–576 46. Packer, A, Clay, K (2000) Soil pathogens and spatial patterns of 63. Van de Peer, Y, De Wachter, R (1994) Treecon for Windows—A seedling mortality in a temperate tree. Nature 404: 278–281 software package for the construction and drawing of evolutionary 47. Packer, A, Clay, K (2003) Soil pathogens and Prunus serotina trees for the Microsoft Windows environment. Comput Appl seedling and sapling growth near conspecific trees. Ecology 84: 108– Biosci 10: 569–570 119 64. Van de Peer, Y, De Wachter, R (1996) Substitution rate calibration 48. Petersen, AB, Rosendahl, S (2000) Phylogeny of the Peronospor- of nucleotide sequence alignments and application to phylogenetic omycetes (Oomycota) based on partial sequences of the large tree construction. Arch Physiol Biochem 104: B53 ribosomal subunit (LSU rDNA). Mycol Res 104: 1295–1303 65. Van de Peer, Y, De Wachter, R (1997) Construction of 49. Riethmu¨ller, A, Voglmayr, H, Go¨ker, M, Weiß, M, Oberwinkler, F evolutionary distance trees with TREECON for Windows: Ac- (2002) Phylogenetic relationships of the downy mildews (Perono- counting for variation in nucleotide substitution rate among sites. sporales) and related groups based on nuclear large subunit Comput Appl Biosci 13: 227–230 ribosomal DNA sequences. Mycologia 94: 834–849 66. Van de Peer, Y, De Wachter, R (1993) Treecon—A software 50. Riethmu¨ller, A, Weiß, M, Oberwinkler, F (1999) Phylogenetic package for the construction and drawing of evolutionary trees. studies of Saprolegniomycetidae and related groups based on Comput Appl Biosci 9: 177–182 nuclear large subunit ribosomal DNA sequences. Can J Bot 77: 67. Van der Auwera, G, Chapelle, S, De Wachter, R (1994) Structure 1790–1800 of the large ribosomal subunit RNA of Phytophthora megasperma, 51. Saitou, N, Nei, M (1987) The neighbor-joining method—A new and phylogeny of the oomycetes. FEBS Lett 338: 133–136 method for reconstructing phylogenetic trees. Mol Biol Evol 4: 68. Van der Auwera, G, Debaere, R, Van de Peer, Y, Derijk, P, Van den 406–425 Broeck, I, De Wachter, R (1995) The phylogeny of the Hyphochy- 52. Sanchez, J, Sanchez-Cara, J, Gallego, E (2000) Suitability of ten triomycota as deduced from ribosomal-RNA sequences of Hypho- plant baits for the rapid detection of pathogenic Pythium species chytrium catenoides. Mol Biol Evol 12: 671–678 in hydroponic crops. Eur J Plant Pathol 106: 209–214 69. Van der Plaats-Niterink, AJ (1981) Monograph of the Genus 53. Schadt, CW, Martin, AP, Lipson, DA, Schmidt, SK (2003) Seasonal Pythium, Vol. Centraalbureau voor Schimmelcultures, Baarn dynamics of previously unknown fungal lineages in tundra soils. 70. van Elsas, JD, Duarte, GF, Keijzer-Wolters, A, Smit, E (2000) Science 301: 1359–1361 Analysis of the dynamics of fungal communities in soil via fungal- 54. Schneider, S, Roessli, D, Excoffier, L (2001) Arlequin ver 2.001: A specific PCR of soil DNA followed by denaturing gradient gel Software for Population Genetics Data Analysis. University of electrophoresis. J Microbiol Methods 43: 133–151 Geneva, Geneva, Switzerland 71. Vandenkoornhuyse, P, Baldauf, SL, Leyval, C, Straczek, J, Young, 55. Shahzad, S, Dick, MW (1990) Effect of different natural baits on JPW (2002) Evolution—Extensive fungal diversity in plant roots. zoospore and oospore production by two Pythium species. Pak J Science 295: 2051 Bot 22: 125–128 72. Verma, BL, Khulbe, D (1983) Achlya flagellata parasitic on wheat 56. Sliwinski, MK, Goodman, RM (2004) Comparison of crenarchaeal seedlings. Curr Sci (Bangalore) 52: 686 consortia inhabiting the rhizosphere of diverse terrestrial plants 73. Voglmayr, H (2003) Phylogenetic relationships of Peronospora and with those in bulk soil in native environments. Appl Environ related genera based on nuclear ribosomal ITS sequences. Mycol Microbiol 70: 1821–1826 Res 107: 1132–1142 57. Smalla, K, Wieland, G, Buchner, A, Zock, A, Parzy, J, Kaiser, S, 74. Watson, AG (1966) Effect of soil fungicide treatments on Roskot, N, Heuer, H, Berg, G (2001) Bulk and rhizosphere soil inoculum potentials of spermosphere fungi and damping-off. bacterial communities studied by denaturing gradient gel electro- NZJ Agric Res 9: 931–955 phoresis: Plant-dependent enrichment and seasonal shifts revealed. 75. Watson, AG (1966) Seasonal variation in inoculum potentials of Appl Environ Microbiol 67: 4742–4751 spermosphere fungi. NZJ Agric Res 9: 956–963 58. Smit, E, Leeflang, P, Glandorf, B, van Elsas, JD, Wernars, K (1999) 76. Webster, RK, Hall, DH (1970) Achlya klebsiana and Pythium Analysis of fungal diversity in the wheat rhizosphere by sequencing species as primary causes of seed rot and seedling disease of rice in of cloned PCR-amplified genes encoding 18S rRNA and temper- California. Phytopathology 60: 964–968