Molecular identification of the turf grass rapid blight pathogen

Mycologia, 97(1), 2005, pp. 160 166. 2005 by The Mycological Society of America, Lawrence, KS 66044-8897 Molecular identification of the turf grass rapid blight pathogen K.D. Craven 1,2 Center for Integrated Fungal Research, Department of Plant Pathology, North Carolina State University, Raleigh, North Carolina 27695 P.D. Peterson 1 Pee Dee Research and Education Center, Clemson University, Florence, South Carolina 29506 D.E. Windham T.K. Mitchell Center for Integrated Fungal Research, Department of Plant Pathology, North Carolina State University, Raleigh, North Carolina 27695 S.B. Martin Pee Dee Research and Education Center, Clemson University, Florence, South Carolina 29506 Abstract: Rapid blight is a newly described disease on turf grasses, primarily found on golf courses using suboptimal water for irrigation purposes. On the basis of shared morphological characteristics, it has been proposed that the rapid blight pathogen belongs to a genus of stramenopiles, Labyrinthula, which had been known to cause disease of marine plants only. We have collected 10 isolates from four species of turf grass in five states and sequenced portions of the SSU (18S) rdna gene from each to provide a definitive taxonomic placement for rapid blight pathogens. We also included sequences from Labyrinthuloides yorkensis, Schizochytrium aggregatum, Aplanochytrium sp., Thraustochytrium striatum, Achlya bisexualis and several nonturf-grass isolates of Labyrinthula. We found that rapid blight isolates indeed are placed firmly within the genus Labyrinthula and that they lack detectable genetic diversity in the 18S rdna region. We propose that the rapid blight pathogens share a recent common ancestor and might have originated from a single, infected population. Key words: cool-season grasses, Labyrinthula, phylogeny, stramenopile Accepted for publication 20 August 2004. 1 These authors contributed equally to this work. 2 Corresponding author. E-mail: kdcraven@ncsu.edu INTRODUCTION The phylum Labyrinthulomycota is a sister group of the Oomycota, Developayella, Hyphochytrium as well as other nonpigmented stramenopiles (Leander and Porter 2001). The Labyrinthulomycota are distinguished by the presence of cell surface organelles, called bothrosomes, that produce an ectoplasmic network through which these organisms move and feed (Perkins 1972, Porter 1987, Leander and Porter 2001). The ectoplasmic network consists of branching and anastomosing filaments capable of absorbing nutrients as well as attaching the organism to substrates (Porter 1987). Other distinguishing morphological characteristics include mitochondria containing tubular cristae and heterokont, biflagellate zoospores (Porter 1987, Patterson 1989, Leander and Porter 2001). Honda et al (1999) suggested the phylum Labyrinthulomycota consisted of two distinct phylogenetic groups, the Thraustochytrid Phylogenetic Group and the Labyrinthula Phylogenetic Group, but phylogenies derived from small subunit (18S) rdna sequences by Leander and Porter (2001) suggest the phylum actually comprises three genetically distinct clades corresponding to three morphological groups, the labyrinthulids, the thraustochytrids and the labyrinthuloids. All isolates of Labyrinthula spp. and Labyrinthula zosterae analyzed by Leander and Porter (2001) were placed within the labyrinthulids. The labyrinthulids consist of a curious group of organisms, both saprobes and pathogens, some of which cause devastating diseases of sea grasses and other marine organisms (Bower 1987; McLean and Porter 1982; Muehlstein 1988, 1991). Labyrinthula species, commonly referred to as the net slime molds, produce spindle-shaped cells that move within anastomized ectoplasmic networks (Martin et al 1983). In 2002 the term rapid blight was used to describe a disease that was first observed affecting Poa annua putting greens at some California golf courses in 1995 (Martin 2002). Since 1995, rapid blight has been diagnosed on Poa annua, Lolium perenne and Poa trivialis (FIG. 1) from more than 100 golf courses in 11 states across the USA. Rapid blight frequently occurs on golf courses with high soil salts (Na and bicarbonates) due primarily to the quality of irrigation water (Martin and Peterson pers comm). 160

CRAVEN ET AL: IDENTITY OF RAPID BLIGHT PATHOGEN 161 On the basis of shared morphological characteristics associated with the size and shape of its spindleshaped vegetative cells, Olsen et al (2003) proposed that the rapid blight pathogen is a member of genus Labyrinthula. Given the quick emergence and increasing incidence of rapid blight disease on golf course turf, a nationwide survey was initiated to collect and characterize the pathogen. The purpose of this study was to conduct a phylogenetic analysis of the 18S rdna genes from the collected isolates alongside those from marine members of Labyrinthulomycota to clarify the taxonomic position of these rapid blight organisms and evaluate the level of nucleotide variability that exists among them. MATERIALS AND METHODS FIG. 1. Typical disease symptoms caused by the rapid blight pathogen (Labyrinthula sp.) on Poa trivialis overseeded putting green. Isolates were obtained from 10 turf grass samples exhibiting symptoms of rapid blight disease (TABLE I) and identity confirmed using light microscopy (FIG. 2). Cultures were maintained on 1% serum seawater agar (Porter 1987). For DNA isolation, all cultures were grown 4 d on solid media. Cells then were harvested by excision of agar blocks into sterile de-ionized water, followed by vigorous agitation to liberate cells and filtration to remove agar blocks, and 5 min centrifugation at 13 000 rpm. DNA was extracted with a microwave miniprep method described previously (Goodwin and Lee 1993). Two regions from the nuclear 18S rdna gene and one from the internal transcribed spacer region (rits region; between the SSU and large subunit [LSU] genes) were amplified using primer pairs SR1R-NS2, NS3-NS4 and ITS1-ITS4 (SR1R from R. Vigalys at http://www.botany.duke.edu/fungi/mycolab/ primers.htm, all others from White et al 1990). PCR reactions were 30 L in volume and contained 15 mm Tris-HCl, 1.5 mm MgCl2, 50 mm KCl, ph 8.0 in the presence of 200 M of each deoxynucleotide triphosphate (datp, dctp, dgtp and dttp; Panvera, Madison, Wisconsin), 200 nm of primers (Integrated DNA Technologies Inc., Coralville, TABLE I. Isolates examined in this study Species Isolate Host Location GenBank Accessions Achlya bisexualis na na na M32705 Thraustochytrium striatum T91-6 Spartina altiniflora Georgia AF265338 Schizochytrium aggregatum T91-7 Polysiphonia sp. Georgia AF265336 Aplanochytrium sp. SC1-1 na na AF348520 Labyrinthuloides yorkensis JEL Ly Zostera marina New Hampshire AF265333 Labyrinthula sp. f Sap 16-1 na na AF348522 Labyrinthula sp. f Spartina sp. na AF265330 Labyrinthula sp. L59 floating plant sample Japan AB095092 Labyrinthula sp. s Zostera marina na AF265332 Labyrinthula sp. AN-1565 na na AB022105 Labyrinthula sp. AZ-1 Poa trivialis L. Arizona CL603045 Labyrinthula sp. AZ-3 Lolium perenne L. Arizona CL603046 Labyrinthula sp. CA-3 Poa annua L. California CL603047 Labyrinthula sp. CA-4 Poa annua L. California CL603048 Labyrinthula sp. CA-5 Poa annua L. California CL603049 Labyrinthula sp. CA-9 na California CL603050 Labyrinthula sp. SC-2 Lolium perenne L. South Carolina CL603051 Labyrinthula sp. TX-1 Poa annua L. Texas CL603052 Labyrinthula sp. UT1-3 Agrostis sp. Utah CL603053 Labyrinthula sp. UT1-4 Poa annua L. Utah CL603054

162 MYCOLOGIA FIG. 2. Vegetative cells of the rapid blight pathogen (Labyrinthula sp.) at 100 magnification. Scale bar 10 m. Iowa), 0.025 U L-1 Taq DNA polymerase (Qiagen, Valencia, California) and 10 ng of genomic DNA. Reactions were performed in a PE Applied Biosystems DNA thermal cycler (Foster City, California), with 30 cycles of 1 min at 95 C, 1 min at 55 C and 1 min at 72 C, followed by a final 5 min step at 72 C. Water blanks were included as negative controls. All amplification products were verified by 0.8 % agarose gel electrophoresis, followed by visualization with ethidium bromide staining and ultraviolet light. The concentration of products was estimated by comparison with a 100 bp quantitative ladder (Panvera). PCR products were cleaned using Qiaquick spin columns (Quiagen Inc., Valencia, California) and sequenced with primers SR1R, NS2, NS3, NS4, ITS1 and ITS4 on a Perkin- Elmer ABI 3700 capillary sequencer following manufacturer protocols. All sequencing reactions were performed with Big Dye Terminator reagents (Perkin-Elmer/ABI, Foster City, California) in a 20 L volume. Both DNA strands were sequenced. Unique gene sequences identified were deposited in GenBank. 18S rdna gene sequences from several Labyrinthula spp., Aplanochytrium sp., Schizochytrium aggregatum, Thraustochytrium striatum and Achyla bisexualis were obtained from GenBank and included in the analyses. (All accession numbers are listed in TABLE I.) Sequences in this study were aligned with the aid of PileUp implemented in SEQWeb version 1.1 with Wisconsin Package version 10 (Genetics Computer Group, Madison, Wisconsin), and the alignment has been submitted to TreeBASE. PileUp parameters were adjusted empirically; a gap penalty of two and a gap extension penalty of zero resulted in robust alignments. Alignments were scrutinized and adjusted manually. For phylogenetic analysis, sequences from both 18S rdna regions were appended manually to create a single contiguous sequence for each isolate. Gene trees were inferred using PAUP* version 4 (Swofford 1998) under both maximum parsimony (MP) and maximum likelihood (ML) criteria. MP employed the branch and bound option for a robust estimate of the optimal tree and character changes were unweighted and unordered, with gaps treated as missing information. The tree root was estimated by outgroup rooting using Achlya bisexualis (a stramenopile from phylum Oomycota). Support for internal nodes of the inferred phylogeny was estimated using the parametric bootstrap method, with 1000 replications under a MP criterion and a branch and bound search option with simple stepwise addition of sequences and tree bisection-reconnection branch swapping. All clades receiving 70% or higher bootstrap values were considered well supported. For the likelihood analysis, parameters including the proportion of invariable sites, nucleotide frequencies and substitution rates, and gamma shape parameter were estimated from the sequence dataset using ML implemented in ModelTest 3.06 (Posada and Crandall 1998) and used as the starting parameters (AIC selected model) for the subsequent analysis. The ML tree was generated by 10 iterations of random sequence addition, followed by tree-bisectionreconnection. Starting branch lengths were obtained using the Rogers-Swofford approximation method implemented in PAUP. RESULTS PCR amplification of 18S rdna from DNA extracted from 10 rapid blight isolates yielded products of the approximate size expected (approx. 600 700 bp

CRAVEN ET AL: IDENTITY OF RAPID BLIGHT PATHOGEN 163 FIG. 3. Single MP tree obtained from a branch and bound search of SSU rdna gene sequences obtained from 10 rapid blight isolates and other representatives of phylum Labyrinthulomycota, and the oomycete Achlya bisexualis (TABLE I). The tree is 494 steps in length; consistency index 0.7068; retention index 0.7329; rescaled consistency index 0.6172. Of 959 total characters in the aligned sequences, 634 were constant, 201 variable characters were parsimony uninformative and 124 were parsimony informative. Alignment gaps were treated as missing information. Tree is outgroup rooted using Achlya bisexualis. Bootstrap values from 1000 replications generated under MP criteria and a branch and bound search option with simple stepwise addition of sequences and tree bisection-reconnection branch swapping are listed above relevant branches. Rapid blight isolates are designated by a two- or three-letter abbreviation corresponding to their state of origin: AZ Arizona, CA California, SC South Carolina, TX Texas and UTI Utah. from primers SR1R and NS2, 500 600 bp from primers NS3 and NS4). Gene sequencing and subsequent appending of the sequences from both regions resulted in approximately 1300 total base pairs per isolate, of which approximately 900 were unambiguously aligned and used in the subsequent analyses. PCR products approximately 400 bp in length from the ITS region were obtained from five rapid blight isolates (TABLE I) using primers ITS1 and ITS4. MP analysis of the appended 18S rdna sequence alignment resulted in a single most parsimonious tree of 494 steps (FIG. 3), with bootstrap values given above well supported branches. The 10 rapid blight isolates group firmly within a clade containing other

164 MYCOLOGIA known Labyrinthula spp., thus confirming their taxonomic placement in this genus. We found it interesting that there is almost no genetic diversity in this ribosomal DNA region among turf-infecting Labyrinthula spp. (FIG. 3). Of the isolates taken from GenBank and included in this analysis, the closest relatives of the turf-infecting Labyrinthula isolates are Labyrinthula sp. L59 and sp. f., with slightly more distant affinity to Labyrinthula species f. Sap 16-1. This group forms a sister clade of Labyrinthula sp. AN- 1565 and sp. s., and collectively they form a larger, highly supported monophyletic clade. Aplanochytrium sp. SC-1 and Labyrinthuloides yorkensis form a well-supported clade adjacent to all Labyrinthula isolates, with Schizochytrium aggregatum lying basal to this entire group. Finally Thraustochytrium striatum and the oomycete Achlya bisexualis (defined as the outgroup) are placed as the most basal lineages. Likelihood ratio testing in ModelTest 3.06 identified the GTR G model of sequence evolution as the most appropriate for our dataset (FIG. 4). The resulting ML tree supports our MP analysis in placing the rapid blight pathogens in the genus Labyrinthula (FIG. 4). In contrast to MP, ML analysis groups Labyrinthula sp. f. Sap 16-1 closest to the marine isolates sp. L59 and sp. f., although on a relatively long branch. All other relationships are the same as MP. To assess whether we could detect greater nucleotide variability among rapid blight Labyrinthula isolates, we sequenced the rits region of five isolates from Arizona and California (data not shown). Although approximately 400 450 bases were analyzed for each, not a single polymorphism was found in this region for any of the five organisms. DISCUSSION Our molecular data confirm the identity of the rapid blight pathogens as members of the labyrinthulid genus Labyrinthula. The inclusion of the pathogens in this genus was suggested by Olsen et al (2003), who isolated fusiform-shaped cells of a size and shape consistent with Labyrinthula from infected Poa trivialis and Lolium perenne (rough bluegrass and perennial ryegrass respectively) and demonstrated a causal role in pathogenicity through completion of Koch s postulates. However many of the morphological characters that typify Labyrinthula also are characteristic of Thraustochytrids and other related stramenopiles, and thus gene sequence data was necessary to provide a definitive taxonomic placement. The nested position of the rapid blight Labyrinthula organisms within a larger clade of marine isolates, together with the more recent reports of turf pathogenesis compared to older reported marine epidemics, suggests that colonization of land plants likely occurred subsequent to marine plant invasion. Of the sequences included here, the rapid blight Labyrinthula isolates are most closely related to Labyrinthula sp. L59 (Kumon et al 2003), Labyrinthula sp. f (Leander and Porter 2001), and Labyrinthula sp. f Sap 16-1, indicating that they might have arisen from one or more of these marine isolates or that they share a recent common ancestor. Of particular interest here is the apparent lack of sequence diversity in this ribosomal gene among the rapid blight Labyrinthula isolates (FIG. 3). The small subunit RNA region is typically suitable for distinguishing between genera and species but often lacks the diversity necessary to distinguish among members of a given species. We chose this region because thus far it has been the only region used to study the systematics of this phylum, thus providing additional sequences for comparison. We analyzed the internal transcribed spacer (ITS) region from five geographically separated rapid blight isolates in search of detectable genetic diversity and found these sequences to be identical. Although the ITS region is often variable enough to discriminate among close relatives, it appears to be quite homogenous among the rapid blight organisms studied here. We are recognizant of the notion that any single gene tree may not accurately reflect the true species tree and currently are pursuing additional gene sequences and rapid blight isolates to further refine the evolutionary relationships within the genus and to evaluate whether any detectable nucleotide polymorphism can be found. If additional sequence data similarly lacks genetic variation, the implication would be a recent common ancestor for the rapid blight pathogens. One possibility is that the turf-infecting Labyrinthula isolates have been spread, apparently quite rapidly, from a single infected population. Given how little we currently understand regarding the life cycle of Labyrinthula and the epidemiology of rapid blight disease, it is premature to speculate on the source of inoculum from which these infections have arisen. It is important to note that the rapid blight Labyrinthula isolates appear only distantly related to the more notorious marine pathogen, Labyrinthula zosterae (data not shown), causative agent of eelgrass wasting disease. Two isolates of L. zosterae originally were included in our analysis, but significant gene sequence divergence made accurate alignment exceedingly difficult. In fact the divergence between L. zosterae and the remaining Labyrinthula isolates was greater than that between the latter and sequences from organisms separated into different genera, such as Labyrinthuloides, Thraustochytrium and Schizochytrium. We subsequently chose to exclude them from

CRAVEN ET AL: IDENTITY OF RAPID BLIGHT PATHOGEN 165 FIG. 4. Tree inferred under a ML criterion with parameters conforming to a GTR G model of sequence evolution. Nucleotide frequencies estimated from a neighbor-joining tree as A 0.3102, T 0.2757, G 0.2413, C 0.1728; proportion of invariable sites 0; gamma shape parameter 0.4768; starting branch lengths obtained by Rogers-Swofford approximation method; starting tree obtained via stepwise addition with a random addition sequence and a tree-bisectionreconnection branch-swapping algorithm. Tree is outgroup rooted using Achlya bisexualis. Likelihood value was ln L 3654.59. Rapid blight isolates are designated as in FIG. 3. our analysis, but our results suggest both that the rapid blight pathogens likely did not arise from L. zosterae and that classification of this latter species might need to be revisited. Before the Olsen et al (2003) report the Labyrinthulomycota had been documented as causing plant disease only on marine species such as Zostera marina (eelgrass) and other sea grasses (Muehlstein et al 1988). Such habitats are characterized by high salinity, suggesting that a saline environment aids Labyrinthula survival. It is perhaps not surprising that pathogenesis of land plants appears associated primarily with golf courses with high soil salts due primarily to poor quality irrigation water. Whether turf grass infections can be reduced if water quality and soil salinity are normalized is a question of epidemiological importance that we currently are addressing. The emergence of rapid blight disease appears to represent an example of conducive cultural practices (in this case the use of unsuitable water for irrigation) that provided Labyrinthula organisms the opportunity to interact with a new group of hosts, coolseason grasses in the subfamily Pooideae. Although several of the marine hosts are given common names including the grass epithet, this is a misnomer in the sense that many of these plants share little taxo-

166 MYCOLOGIA nomic affinity with true grasses (family Poaceae). Host information was not available for many of the organisms for which we extracted sequence data from GenBank, but it is noteworthy that Labyrinthula sp. f (one of two marine isolates most closely related to the rapid blight isolates) infects Spartina alterniflora, a true grass. It is possible that Labyrinthula isolates infecting true marine grasses are a likely source from which turf infections arise. The role of salinity in the disease cycle of marine Labyrinthula isolates is unclear although it might be important in dissemination between susceptible hosts, when the organism is less buffered from the external environment by host tissues. Adaptation to land plants that themselves do not require high salinity raises the specter of a similar loss of dependence in the rapid blight pathogens. ACKNOWLEDGMENTS We acknowledge the cooperation of Julie Benson and other members of the Fungal Genomics Lab at North Carolina State University for insightful comments and assistance in gene sequencing. We also wish to thank Ignazio Carbone for critical review of the data. We acknowledge partial financial support of this project from a grant from the United States Golf Association. LITERATURE CITED Bower SM. 1987. Labyrinthuloides haliotidis n. sp. (Protozoa: Labyrinthumorpha), a pathogenic parasite of small juvenile abalone in a British Columbia mariculture facility. Can. J. Zool. 65:1996 2007. Goodwin DC, Lee SB. 1993. Rapid, microwave mini-prep of total genomic DNA from fungi, plants, protists and animals for PCR. Biotechniques 15:438 444. Honda D, Yokochi T, Nakahara T, Raghukumar S, Nakagiri A, Schaumann K, Higashibara T. 1999. Molecular phylogeny of labyrinthulids and thraustochytrids based on the sequencing of 18S ribosomal RNA gene. Eur. J. Protistol 46:637 647. Kumon Y, Yokoyama R, Yokochi T, Honda D, Nakahara T. 2003. A new labyrinthulid isolate, which solely produces n-6 docosapentaenoic acid. Appl. Microbiol. Biotechnol. 63:22 28. Leander CA, Porter D. 2001. The Labyrinthulomycota is comprised of three distinct lineages. Mycologia 93:459 464. Martin GW, Alexopoulis CJ, Farr ML. 1983. The Genera of Myxomycetes. Iowa City, Iowa: University of Iowa Press. Martin SB, Stowell LJ, Gelernter WD, Alderman SC. 2002. Rapid blight: a new disease of cool-season turf grasses. Phytopathology 92:S52. (Abstract) McLean N, Porter D. 1982. The yellow-spot disease of Tritonia diomsdea Bergh, 1894 (Mollusca: Gastropoda: Nudibranchia): Encapsulation of the thraustochytriaceous parasite by host amoebocytes. J. Parasitol 68:243 252. Muehlstein LK, Porter D, Short FT. 1988. Labyrinthula sp., a marine slime mold producing the symptoms of wasting disease in eelgrass, Zostera marina. Mar. Biol 99: 465 472. Muehlstein LK, Porter D, Short FT. 1991. Labyrinthula zosterae sp. Nov., the causative agent of wasting disease of eelgrass, Zostera marina. Mycologia 83:180 191. Olsen MW, Bigelow DM, Gilbertson RL, Stowell LJ, Gelernter WD. 2003. First report of a Labyrinthula sp. causing rapid blight disease of rough bluegrass and perennial ryegrass. Plant Disease 87:1267. Patterson DJ. 1989. Stramenopiles: chromophytes from a protistan perspective. In: Green JC, Leadbeater BSC, Diver WI, eds. The chromophyte algae: problems and perspectives. Systemat Assoc Special Vol 38. Oxford: Clarendon Press. p 357 379. Perkins FO. 1972. The ultrastructure of holdfasts, rhizoids and slime tracks in thraustochytriaceous fungi and Labyrinthula spp. Archiv für Mikrobiologie 84:95 118. Porter D. 1987. Labyrinthulomycetes. In: Fuller MS, Jaworski, A, eds. Zoosporic fungi in teaching and research. Athens, Georgia: Southeastern Publishing Corp. p 110 111. Posada D, Crandall KA. 1998. ModelTest: testing the model of DNA substitution. Bioinformatics 14 (9):817 818. Swofford DL. 1998. PAUP*. Phylogenetic Analysis Using Parsimony (*and other methods). Sunderland, Massachusetts: Sinauer Associates Inc. White TJ, Bruns T, Lee S, Taylor JW. 1990. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: Innis MA, Gelfand DH, Sninsky JJ, White TJ, eds. PCR protocols: a guide to methods and applications. New York: Academic Press Inc. p 315 322.