Molecular identification of the turf grass rapid blight pathogen

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Molecular identification of the turf grass rapid blight pathogen

K.D. Craven1,2

Center for Integrated Fungal Research, Department of Plant Pathology, North Carolina State University, Raleigh, North Carolina 27695

P.D. Peterson1

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 ba-sis of shared morphological characteristics, it has been proposed that the rapid blight pathogen be-longs 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 por-tions of the SSU (18S) rDNA gene from each to pro-vide a definitive taxonomic placement for rapid blight pathogens. We also included sequences from Labyrinthuloides yorkensis, Schizochytrium aggregatum, Aplanochytriumsp.,Thraustochytrium striatum, Achlya bisexualis and several nonturf-grass isolates of Laby-rinthula. We found that rapid blight isolates indeed are placed firmly within the genus Labyrinthulaand that they lack detectable genetic diversity in the 18S rDNA region. We propose that the rapid blight path-ogens share a recent common ancestor and might have originated from a single, infected population.

Key words: cool-season grasses,Labyrinthula, phy-logeny, stramenopile

Accepted for publication 20 August 2004. 1_{These authors contributed equally to this work.} 2_{Corresponding author. E-mail: [email protected]}

INTRODUCTION

The phylum Labyrinthulomycota is a sister group of the Oomycota,Developayella, Hyphochytriumas well as other nonpigmented stramenopiles (Leander and Porter 2001). The Labyrinthulomycota are distin-guished by the presence of cell surface organelles, called bothrosomes, that produce an ectoplasmic net-work through which these organisms move and feed (Perkins 1972, Porter 1987, Leander and Porter 2001). The ectoplasmic network consists of branch-ing and anastomosbranch-ing filaments capable of absorbbranch-ing nutrients as well as attaching the organism to sub-strates (Porter 1987). Other distinguishing morpho-logical characteristics include mitochondria contain-ing tubular cristae and heterokont, biflagellate zoo-spores (Porter 1987, Patterson 1989, Leander and Porter 2001).

Honda et al (1999) suggested the phylum Labyrin-thulomycota consisted of two distinct phylogenetic groups, the Thraustochytrid Phylogenetic Group and the Labyrinthula Phylogenetic Group, but phyloge-nies derived from small subunit (18S) rDNA sequenc-es by Leander and Porter (2001) suggsequenc-est the phylum actually comprises three genetically distinct clades corresponding to three morphological groups, the la-byrinthulids, the thraustochytrids and the labyrinthu-loids. All isolates ofLabyrinthulaspp. and Labyrinthu-la 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 anas-tomized ectoplasmic networks (Martin et al 1983).

In 2002 the term ‘‘rapid blight’’ was used to de-scribe a disease that was first observed affectingPoa annuaputting greens at some California golf courses in 1995 (Martin 2002). Since 1995, rapid blight has been diagnosed on Poa annua, Lolium perenneand 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 irriga-tion water (Martin and Peterson pers comm).

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FIG. 1. Typical disease symptoms caused by the rapid blight pathogen (Labyrinthula sp.) on Poa trivialis over-seeded putting green.

TABLEI. 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 Polysiphoniasp. Georgia AF265336

Aplanochytriumsp. SC1-1 na na AF348520

Labyrinthuloides yorkensis JEL Ly Zostera marina New Hampshire AF265333

Labyrinthulasp. f Sap 16-1 na na AF348522

Labyrinthulasp. f Spartinasp. na AF265330

Labyrinthulasp. L59 floating plant sample Japan AB095092

Labyrinthulasp. s Zostera marina na AF265332

Labyrinthulasp. AN-1565 na na AB022105

Labyrinthulasp. AZ-1 Poa trivialisL. Arizona CL603045

Labyrinthulasp. AZ-3 Lolium perenneL. Arizona CL603046

Labyrinthulasp. CA-3 Poa annuaL. California CL603047

Labyrinthulasp. CA-9 na California CL603050

Labyrinthulasp. SC-2 Lolium perenneL. South Carolina CL603051

Labyrinthulasp. TX-1 Poa annuaL. Texas CL603052

Labyrinthulasp. UT1-3 Agrostissp. Utah CL603053

Labyrinthulasp. UT1-4 Poa annuaL. Utah CL603054

On the basis of shared morphological characteris-tics associated with the size and shape of its spindle-shaped vegetative cells, Olsen et al (2003) proposed that the rapid blight pathogen is a member of genus Labyrinthula. Given the quick emergence and in-creasing incidence of rapid blight disease on golf course turf, a nationwide survey was initiated to

col-lect 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 Labyrin-thulomycota to clarify the taxonomic position of these rapid blight organisms and evaluate the level of nucleotide variability that exists among them.

MATERIALS AND METHODS

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 prim-er 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 reac-tions were 30mL in volume and contained 15 mM Tris-HCl, 1.5 mM MgCl2, 50 mM KCl, pH 8.0 in the presence of 200

mM of each deoxynucleotide triphosphate (dATP, dCTP, dGTP and dTTP; Panvera, Madison, Wisconsin), 200 nM of primers (Integrated DNA Technologies Inc., Coralville,

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FIG. 2. Vegetative cells of the rapid blight pathogen (Labyrinthulasp.) at 1003magnification. Scale bar510mm. Iowa), 0.025 UmL-1 Taq DNA polymerase (Qiagen,

Valen-cia, 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 con-trols. All amplification products were verified by 0.8 % aga-rose gel electrophoresis, followed by visualization with eth-idium bromide staining and ultraviolet light. The concen-tration 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 20mL volume. Both DNA strands were sequenced. Unique gene sequences identified were depos-ited in GenBank. 18S rDNA gene sequences from several

Labyrinthulaspp.,Aplanochytriumsp.,Schizochytrium aggre-gatum, Thraustochytrium striatumandAchyla bisexualiswere 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 re-sulted 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 like-lihood (ML) criteria. MP employed the branch and bound option for a robust estimate of the optimal tree and char-acter changes were unweighted and unordered, with gaps treated as missing information. The tree root was estimated by outgroup rooting usingAchlya 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 cri-terion and a branch and bound search option with simple stepwise addition of sequences and tree bisection-reconnec-tion branch swapping. All clades receiving 70% or higher bootstrap values were considered well supported.

For the likelihood analysis, parameters including the pro-portion of invariable sites, nucleotide frequencies and sub-stitution 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 subse-quent analysis. The ML tree was generated by 10 iterations of random sequence addition, followed by tree-bisection-reconnection. 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

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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 oomyceteAchlya bisexualis(TABLEI). The tree is 494 steps in length; consistency index50.7068; retention index50.7329; rescaled consistency index50.6172. 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 usingAchlya 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: AZ5Arizona, CA5California, SC5South Carolina, TX5Texas and UTI5Utah.

from primers SR1R and NS2, 500–600 bp from prim-ers NS3 and NS4). Gene sequencing and subsequent appending of the sequences from both regions re-sulted in approximately 1300 total base pairs per iso-late, of which approximately 900 were unambiguous-ly aligned and used in the subsequent anaunambiguous-lyses. PCR products approximately 400 bp in length from the

ITS region were obtained from five rapid blight iso-lates (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

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known Labyrinthula spp., thus confirming their tax-onomic placement in this genus. We found it inter-esting that there is almost no genetic diversity in this ribosomal DNA region among turf-infecting Labyrin-thula spp. (FIG. 3). Of the isolates taken from

GenBank and included in this analysis, the closest relatives of the turf-infectingLabyrinthulaisolates are Labyrinthulasp. L59 and sp. f., with slightly more dis-tant affinity toLabyrinthula 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. Aplanochy-trium sp. SC-1 and Labyrinthuloides yorkensisform a well-supported clade adjacent to allLabyrinthula iso-lates, 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 identi-fied the ‘‘GTR1G’’ 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 genusLabyrinthula (FIG. 4). In contrast to MP, ML analysis groups La-byrinthulasp. 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 nucleo-tide variability among rapid blight Labyrinthula iso-lates, we sequenced the rITS region of five isolates from Arizona and California (data not shown). Al-though 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 ge-nus 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 con-sistent 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 pos-tulates. However many of the morphological charac-ters that typifyLabyrinthulaalso are characteristic of Thraustochytrids and other related stramenopiles, and thus gene sequence data was necessary to pro-vide a definitive taxonomic placement.

The nested position of the rapid blight Labyrinthu-laorganisms within a larger clade of marine isolates, together with the more recent reports of turf path-ogenesis compared to older reported marine

epi-demics, suggests that colonization of land plants like-ly occurred subsequent to marine plant invasion. Of the sequences included here, the rapid blight Laby-rinthula isolates are most closely related to Labyrin-thulasp. L59 (Kumon et al 2003),Labyrinthulasp. 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 blightLabyrinthula isolates (FIG. 3). The small

subunit RNA region is typically suitable for distin-guishing between genera and species but often lacks the diversity necessary to distinguish among mem-bers 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 geograph-ically separated rapid blight isolates in search of de-tectable genetic diversity and found these sequences to be identical. Although the ITS region is often var-iable 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 accu-rately reflect the true species tree and currently are pursuing additional gene sequences and rapid blight isolates to further refine the evolutionary relation-ships within the genus and to evaluate whether any detectable nucleotide polymorphism can be found. If additional sequence data similarly lacks genetic var-iation, the implication would be a recent common ancestor for the rapid blight pathogens. One possi-bility is that the turf-infecting Labyrinthula isolates have been spread, apparently quite rapidly, from a single infected population. Given how little we cur-rently understand regarding the life cycle of Labyrin-thula and the epidemiology of rapid blight disease, it is premature to speculate on the source of inocu-lum from which these infections have arisen.

It is important to note that the rapid blight Laby-rinthula isolates appear only distantly related to the more notorious marine pathogen, Labyrinthula zos-terae (data not shown), causative agent of eelgrass wasting disease. Two isolates of L. zosteraeoriginally were included in our analysis, but significant gene sequence divergence made accurate alignment ex-ceedingly difficult. In fact the divergence betweenL. 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 Schizochy-trium. We subsequently chose to exclude them from

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FIG. 4. Tree inferred under a ML criterion with parameters conforming to a ‘‘GTR1G’’ model of sequence evolution. Nucleotide frequencies estimated from a neighbor-joining tree as A5 0.3102, T5 0.2757, G 50.2413, C 50.1728; pro-portion of invariable sites 5 0; gamma shape parameter 5 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-bisection-reconnection branch-swapping algorithm. Tree is outgroup rooted usingAchlya bisexualis. Likelihood value was2ln L5 3654.59. Rapid blight isolates are designated as in FIG. 3.

our analysis, but our results suggest both that the rap-id blight pathogens likely drap-id not arise from L. zos-teraeand that classification of this latter species might need to be revisited.

Before the Olsen et al (2003) report the Labyrin-thulomycota had been documented as causing plant disease only on marine species such asZostera marina (eelgrass) and other sea grasses (Muehlstein et al 1988). Such habitats are characterized by high salin-ity, suggesting that a saline environment aids Labyrin-thulasurvival. It is perhaps not surprising that path-ogenesis of land plants appears associated primarily with golf courses with high soil salts due primarily to

poor quality irrigation water. Whether turf grass in-fections can be reduced if water quality and soil sa-linity 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 irriga-tion) that provided Labyrinthula organisms the op-portunity to interact with a new group of hosts, cool-season 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

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taxo-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 thatLabyrinthula sp. f (one of two marine isolates most closely related to the rapid blight isolates) infects Spartina alterni-flora,a true grass. It is possible thatLabyrinthula iso-lates infecting true marine grasses are a likely source from which turf infections arise. The role of salinity in the disease cycle of marineLabyrinthulaisolates is unclear although it might be important in dissemi-nation between susceptible hosts, when the organism is less buffered from the external environment by host tissues. Adaptation to land plants that them-selves 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 fi-nancial support of this project from a grant from the Unit-ed States Golf Association.

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