Microbial Ecology
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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