In Press at Mycologia, preliminary version published on August 6, 2013 as doi:10.3852/13-010

Short title:

Oomycetes baited from streams in Tennessee 2010–2012

Sandesh K. Shrestha

Yuxin Zhou

Kurt Lamour1

Department of Entomology and , University of Tennessee, Knoxville, Tennessee

37996

Abstract: Sixteen streams in middle and eastern Tennessee were surveyed for the sudden oak-

death pathogen ramorum 2010–2012. Surveys were conducted in the spring and

fall using healthy Rhododendron leaves, and a total of 354 isolates were recovered.

Sequence analysis of the ITS region provisionally identified 151 Phytophthora, 200 Pythium,

two Halophytophthora and one Phytopythium. These include six Phytophthora species (P.

cryptogea, P. hydropathica, P. irrigata, P. gonapodyides, P. lacustris, P. polonica), members of the P. citricola species complex, five unknown Phytophthora species, 11 Pythium species (P. helicoides, P. diclinum, P. litorale, P. senticosum, P. undulatum, P. vexans, P. citrinum, P. apleroticum, P. chamaihyphon, P. montanum, P. pyrilobum), three unknown Pythium species,

Halophytophthora batemanensis, and one Phytopythium isolate. The biology and implications are discussed.

Key words: environmental samples, Oomycetes, species diversity

INTRODUCTION

Oomycetes thrive in wet environments and their evolutionary relatives (e.g. diatoms and brown

algae) are aquatic (Baldauf et al. 2000, Garbelotto et al. 2001, Lamour and Kamoun 2009). Many

oomycete genera contain species attacking plants within managed and wild settings, and these

Copyright 2013 by The Mycological Society of America.

organisms produce distinctive for survival and spread. Spores include thick-walled sexual oospores and asexual chlamydospores, which are able to survive extended periods, and shorter- lived asexual sporangia, which can release swimming zoospores. Sporangia can be deciduous and serve as a primary method of dispersal or release swimming zoospores (Erwin and Ribeiro

1996). Swimming zoospores can survive extended periods in free water allowing for spread via irrigation, drainage and natural waterways (Ghimire et al. 2009).

Oomycete identification can be difficult based solely on morphological characters and the use of molecular genetic approaches is common (Oudemans and Coffey 1991, Brasier et al.

1993, Erwin and Ribeiro 1996, Cooke and Duncan 1997, Lamour and Kamoun 2009). The most frequently used genetic loci are the internal transcribed spacers (ITS1, ITS2) between the 18S and 28S ribosomal RNA genes (Lee and Taylor 1992, Cooke and Duncan 1997, Cooke et al.

2000;). Although the ITS region is useful in identifying many well described species, it may not provide sufficient resolution to clearly identify members of evolutionarily distinct, yet closely related, species complexes (e.g. P. citricola species complex) (Jung and Burgess 2009). For closely related species it may be necessary to assess multiple genes as well as morphological characters.

Ornamental and wild species of Rhododendron are susceptible to many Phytophthora species including P. cinnamomi, P. cactorum, P. citricola, P. lateralis, P. cryptogea, P. citrophthora, P. gonapodyides, P. megasperma and the sudden oak-death (SOD) pathogen P. ramorum. In recent years Rhododendron leaves have been used extensively to monitor waterways for P. ramorum, leading to the recovery of P. ramorum in streams in California,

Oregon and Washington (Hoitink and Schmitthenner 1974, Werres et al. 2001, Oak et al. 2008).

Surveys for SOD in USA are ongoing at nurseries and natural areas considered potentially high

risk, including Tennessee (Rizzo et al. 2002, Donahoo and Lamour 2008, Oak et al. 2008, Sutton et al. 2009, Hulvey et al. 2010). In Tennessee, surveys have been conducted since 2004 and P. ramorum was recovered from nurseries in 2004 and 2005 (California 2012). P. ramorum also has been isolated from nurseries in 20 additional states (California 2012). In 2004 and 2005, seven species of Phytophthora (P. cactorum, P. citricola, P. citrophthora, P. nicotianae, P. palmivora,

P. tropicalis and the new species P. foliorum) were recovered from nurseries in Tennessee

(Donahoo and Lamour 2008). Subsequent surveys identified five Phytophthora spp. (P. citrophthora, P. citricola, P. nicotianae, P. syringae, P. hydropathica) from statewide nurseries and four species (P. citrophthora, P. citricola, P. irrigata, P. hydropathica) from streams of eastern Tennessee (Hulvey et al. 2010).

Tennessee is one of the largest producers of oak trees and has significant P. ramorum- susceptible natural areas (e.g. Smoky Mountains National Park) important to tourism and the

statewide economy (Forest product extension 2012). In 2009, forests contributed 4% to the $21 billion Tennessee economy (Menard et al. 2011). Although P. ramorum has been found in

Tennessee nurseries, it has not been recovered from natural settings and here we report species

diversity for oomycetes recovered while surveying streams for P. ramorum in middle and eastern

Tennessee 2010–2012.

MATERIALS AND METHODS

Sampling and pathogen isolation from streams.—Stream sites were selected in watersheds containing plant

nurseries (FIG. 1). In general, the streams drain agricultural and wild areas and to our knowledge none of the streams are directly connected. Baiting was conducted during spring and fall 2010–2012. Asymptomatic healthy mature

Rhododendron maximum leaves were collected in the Smoky Mountains and stored at 5 C 1–3 wk before baiting sessions. Baits were deployed in a mesh bag and anchored by lines to the shore and a weight to hold the bags in place. Two bags were placed at each location with four leaves in each bag. Each baiting session was 7–10 d, with three baiting sessions in the spring and fall. After deployment, leaves were washed in tap water and four leaves were

selected randomly for tissue isolation. The other four leaves were sent to the Plant Disease Diagnostic Lab at

Pennsylvania Department of Agriculture for genetic testing for P. ramorum. The leaves processed in Tennessee were inspected for water-soaked lesions. If lesions were visible, a single lesion from each leaf was chosen and a 10 mm leaf disks containing both healthy tissue and necrotic lesions was transferred to dilute V8-PARP + H agar (40 mL V8 juice, 3 g CaCO3, 16 g Bacto agar, 960 mL water amended with 25 ppm pimaricin (Sigma Aldrich, St Louis,

Missouri), 100 ppm ampicillin (Fisher Scientific, Pittsburg, Pennsylvania), 25 ppm rifampicin (Sigma Aldrich, St

Louis, Missouri), 25 ppm pentachloronitrobenzene (Fisher Scientific, Pittsburg, Pennsylvania) and 71.4 mg/L

Hymexazol (Bayer Corp., Pittsburgh, Pennsylvania) (Hulvey et al. 2010). After incubation 1–3 d, nonseptate mycelium typical of oomycetes was transferred onto water agar and following a few days' growth, a single hyphal tip transferred to full strength V8-PARP agar.

Long term storage, DNA extraction and ITS amplification.—For long term storage, 3–4 plugs (7 mm diam) of actively expanding mycelium were transferred into 2 mL screw-cap tubes preloaded with 1 mL sterilized water and

3–4 sterilized hemp seeds and stored at room temperature. Mycelium was produced and genomic DNA extracted according to the protocol of Lamour and Finley (Lamour and Finley 2006).

The ITS region was PCR amplified with the universal primers ITS5

(5′GGAAGTAAAAGTCGTAACAAGG3′) and ITS4 (5′TCCTCCGCTTATTGATATGC 3′) (White et al. 1990).

The PCR reaction consisted of a 30 µL final volume of 3 µL 10× buffer, 1.2 µL dNTPs at 5 mM, 1.5 µL DNA at

50–100 ng/µL, 1 µL TAQ polymerase and 0.1 µL primers at 100 mM conc. The PCR cycling parameters were: initial denaturation at 94 C for 2 min followed by 29 cycles of denaturation at 94 C for 20 s, annealing at 55 C for 25 s, extension at 72 C for 1 min and final extension at 72 C for 10 min. PCR products were treated to remove excess primers and dNTPs with ExoSAP-IT reagent (USB Corp., Cleveland, Ohio). The reagent was added at the rate of 2

µL per 5 µL PCR product and treated at 37 C for 15 min, followed by 80 C for 15 min (Dugan et al. 2002, Bell

2008). Both ITS4 and ITS5 primers were used for sequencing at the University of Iowa DNA sequencing facility

(Iowa City, Iowa). Sequences were processed and assembled with CodonCode Aligner 3.7.1 software (CodonCode,

Dedham, Massachusetts). Sequences were trimmed by removing ends with > 2 bases with a PHRED score > 20 in a

40-bp window. Resulting contigs with complete ITS1, 5.8s and ITS2 regions were searched using the National

Center for Biotechnology Information (NCBI) GenBank nonredundant nucleotide database and the curated

Phytophthora databases PhytophthoraDB (http://www.phytophthoradb.org/) as of 1 Dec 2012. Sequences matching

known species were determined with coverage and identity and a “best hit” NCBI accession recorded. Isolates were identified as “unknown” if there was < 99% identity to a previously described species or if the sequence matched a previously reported unknown species. For the putative unknowns, the most similar sequence in GenBank was recorded.

RESULTS

A total of 354 isolates were recovered 2010–2012. The known and unknown species are cited

(TABLES I, II). These include six known Phytophthora species (P. cryptogea, P. hydropathica, P.

irrigata, P. gonapodyides, P. lacustris, P. polonica), members of the P. citricola species

complex, five unknown Phytophthora species (designated Phytophthora “Pgchlamydo”, Phy.

Unk1, Phy. Unk2, Phy. Unk3, Phy. Unk4), 11 known Pythium species (P. citrinum, P.

helicoides, P. diclinum, P. litorale, P. senticosum, P. undulatum, P. vexans, P. apleroticum,

P. chamaihyphon, P. montanum, P. pyrilobum), three unknown Pythium species (designated

Py. Unk1, Py. Unk2 and Py. Unk3), Halophytophthora batemanensis and one isolate belonging

to the newly described genus Phytopythium (TABLE I) (Cooke et al. 2000, Kageyama et al. 2002,

Abdelzaher 2004, Paul 2004, Shafizadeh and Kavanagh 2005, Zeng et al. 2005, Belbahri et al.

2006, Nechwatal and Mendgen 2006, Villa et al. 2006, Hong et al. 2008a, Senda et al. 2009,

Hong et al. 2010, Robideau et al. 2011).

The seven isolates assigned to the P. citricola species complex all had identical rDNA

regions that differ at two loci from P. citricola s. str. (NCBI accession number FJ560913). The

two loci (positions 153, 697, counting from the first base of the ITS1 for P. citricola s. str.) are

clearly heterozygous with overlapping high quality electropherogram peaks in both forward and

reverse sequences (Data not shown). Position 153 is a C/T heterozygote and is one of three loci

that differentiate P. citricola (position 153 = T) from the more recently described P. plurivora

(position 153 = C) (Jung and Burgess 2009). Position 697 is a G/A heterozygote and is one of

three loci that differentiate P. citricola I (position 697 = A) and P. citricola III (position 697 =

A) from P. citricola (position 697 = G).

Phytophthora Unk1 has been recovered from forest soil and stream baiting in northwestern Yunnan province, China (JQ755232.1). Phytophthora Unk2 has been recovered from stream water in South Africa (GU799642.1). Phytophthora Unk3 was recovered from stream-baiting in West Virginia and Ohio (EU644720.1). Phytophthora Unk4 is similar to one of two new homothallic nonpapillate species of Phytophthora described from irrigation reservoirs and natural waterways in Virginia, USA (FJ666127.1). Phytophthora Unk4 was similar (> 97% maximum identity) to the provisional species Phytophthora “aquimorbida”. All three unknown

Pythium species had no significant similarity to any known Pythium species (TABLE I). Most

species were found in both the spring and the fall (TABLE II).

DISCUSSION

Forests and natural areas are an important part of the economy of Tennessee. In 2008, Tennessee

produced 881 000 000 board feet of hardwood lumber and the forested state park system is

extensive and highly valuable (Menard et al. 2011). Our objective was to monitor streams in

middle and eastern Tennessee for P. ramorum and to identify culturable oomycetes recovered

during the study.

P. cryptogea was the most frequently occurring Phytophthora species; present in 15 out

of 16 locations in middle and eastern Tennessee (TABLE I). P. cryptogea was recovered in previous Tennessee surveys and also from irrigation water in Virginia and reservoirs in Germany

(Themann et al. 2002, Bush et al. 2003, Donahoo and Lamour 2008, Hulvey et al. 2010). P. cryptogea was more common in the fall when the temperatures were cooler (TABLE II). P.

cryptogea causes minor root rot of Rhododendron (Hoitink and Schmitthenner 1974), but the interaction with oak trees is unknown (Balcì and Halmschlager 2003).

Two other frequent Phytophthora species were P. irrigata and P. hydropathica. P. irrigata has been recovered from streams, rivers and irrigation reservoirs at diverse locations including Tennessee (Hong et al. 2008a, b; Hulvey et al. 2010; Ghimire et al. 2011).

Experimental inoculations indicated P. irrigata is pathogenic to azaleas and some vegetable crops including pepper, tomato and eggplant (Hong et al. 2008b); however, damage has not been reported under field conditions. P. hydropathica was isolated from irrigation reservoirs and first described as a new species in 2010 (Hong et al. 2010, Ghimire et al. 2011). P. hydropathica has been isolated from watersheds and nurseries of eastern Tennessee (Hulvey et al. 2010). P. hydropathica is an important pathogen of various ornamental species causing leaf blight and leaf

necrosis of Rhododendron and root rot of Kalmia (mountain laurel) (Hong et al. 2008b, Hong et

al. 2010). Because P. hydropathica is widespread in Tennessee it might pose a threat to the

nursery ornamentals and forest hosts.

Members of the P. citricola species complex were recovered from five locations and

members of this group have been found in irrigation water (MacDonald et al. 1994, Hong and

Moorman 2005, Donahoo and Lamour 2008, Hulvey et al. 2010). Heterozygosity at two key loci

in the ribosomal DNA repeat may indicate interspecific hybridization for isolates occurring in

Tennessee and further sequencing and morphological characterizations are necessary to properly

ID these isolates (Hurtado-Gonzales et al. 2009). Members of the P. citricola species complex

have a wide host range that includes economically important fruit trees, such as avocado, walnut,

and , and forest trees (Bhat and Browne 2007). Studies of P. citricola from Tennessee

indicate isolates are genetically diverse, are likely moved via nursery stocks and might be a

threat to forest trees (Donahoo and Lamour 2008, Hulvey et al. 2010). P. gonapodyides, recovered from three locations, has been recovered from stream, reservoir, soil and stem samples of declining oak stands (Jung et al. 1996, Balcì and Halmschlager 2003, Bush et al. 2003,

Ghimire et al. 2011) and is pathogenic to conifers (Hamm and Hansen 1982) and many ornamentals (Erwin and Ribeiro 1996). A similar survey of irrigation reservoirs in Virginia reports nine species of Phytophthora and hundreds of unknown Phytophthora isolates (Bush et al. 2006; Hong et al. 2008a). Some of the unknown Phytophthora isolates described here were reported from Tennessee. Phytophthora “Pgchlamydo” was recovered from ornamentals plants, forest watershed and stream-baiting (Moralejo et al. 2009; Reeser et al. 2011a, b), and artificial inoculation of Rhododendron indicates it can cause leaf lesions (Schwingle and Blanchette

2008). Leaf spot caused by Phytophthora “Pgchlamydo” on evergreen nursery stock has been reported in California (Blomquist et al. 2012). Phytophthora lacustris, recovered from two locations, is the formal taxonomic description of the provisional Phytophthora species referred to as P. taxon Salixsoil (Brasier et al. 2003). It has been reported from forest ecosystems (soil) or rivers and lakes in Europe, USA, Western Australia and China. Studies indicated that in addition to the breakdown of plant detritus it might be an opportunistic fine-root pathogen in flooding habitats (Nechwatal et al. 2012). P. polonica, found at two locations, has been recovered from soil around declining trees and in laboratory tests was slightly pathogenic to alder twigs but not pathogenic to trunks of several tree species (Belbahri et al. 2006). Halophytophthora batemanensis shares many attributes with Phytophthora and only recently was segregated from the latter genus (Ho and Jong 1990, Erwin and Ribeiro 1996, Goker et al. 2007). Finally one isolate of the newly described genus Phytopythium was recovered (Bala et al. 2010).

This is the first report of species diversity for Pythium in Tennessee. The P. ramorum selective media contains the chemical Hymexazol, which is inhibitory to some Pythium species

(Erwin and Ribeiro 1996). This inhibitory effect might have aided our isolation efforts in cases where mycelial growth is slowed but not fully blocked, although additional research is needed to fully characterize the effects. More work is needed to better understand the diversity, distribution and roles played by Pythium in Tennessee natural areas. Although P. ramorum was not found during this survey period, the identification of diverse known and unknown Phytophthora and

Pythium provides a useful baseline for species diversity and may be useful if any of these species poses a threat to plants in the environment or to plants produced commercially. And finally additional research is needed to formally describe the unknown species and to test their host range, particularly within Tennessee.

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LEGENDS

FIG. 1. Sixteen locations in Tennessee surveyed for Phytophthora ramorum by baiting with healthy Rhododendron leaves.

FOOTNOTES

Submitted 8 Jan 2013; accepted for publication 16 Apr 2013.

1Corresponding author. E-mail: [email protected]

TABLE I. Data for Phytophthora, Pythium, Halophytophthora and Phytopythium species recovered from middle and eastern Tennessee 2010–2012 Name NCBI taxonomy Number Query Maximum Location ID/accession of coverage identity numbers isolates Phytophthora species P. cryptogea 4786 87 99 99 TN1, TN2, TN3,TN4, TN5, TN6, TN7, TN8, TN9, TN10, TN11, TN12,TN13, TN15, TN16 P. hydropathica 565288 21 99 99 TN1, TN2, TN4, TN7,TN10, TN12 P. irrigata 565287 7 95 100 TN2, TN3, TN4, TN7, TN8, TN9 P. gonapodyides 78237 5 100 97 TN1, TN2, TN9 P. lacustris 1190534 4 97 100 TN2, TN6 P. polonica 374175 3 100 100 TN6, TN7 Phy Unk1 JQ755232.1 7 100 99 TN1, TN2, TN7, TN9 Phy Unk2 GU799642.1 3 100 100 TN5 Phy Unk3 EU644720.1 1 100 99 TN1 Phy Unk4 FJ666127.1 1 93 97 TN7 P. “Pgchlamydo” 479473 5 100 99 TN7, TN10, TN13 P. citricola 53984 7 100 99 TN2, TN3, TN6, complex TN9,TN10 Pythium species P. citrinum 221743 7 98 94 TN1, TN2, TN3, TN5, TN15 P. diclinum 156628 24 100 94 TN1, TN2, TN3, TN5, TN6, TN7, TN13 P. helicoides 126844 2 99 99 TN1, TN4 P. litorale 340183 112 99 99 TN1, TN2, TN3, TN4, TN5, TN6, TN7, TN8, TN9, TN10, TN11, TN12, TN13, TN14, TN15, TN16 P. senticosum 643143 1 100 99 TN5 P. undulatum 127444 4 100 99 TN2, TN4, TN5 P. vexans 42099 1 99 99 TN7 P. apleroticum 289600 1 100 99 TN3 P. chamaihyphon 82933 1 100 100 TN11 P. montanum 214887 1 100 99 TN13 P. pyrilobum 289602 29999TN1 Py Unk1 HQ261735.1 34 100 96 TN1, TN2, TN3, TN4, TN5, TN6, TN7, TN8, TN9, TN10 Py Unk2 DQ232768.1 4 100 100 TN4, TN7, TN9, TN13 Py Unk3 HQ261735.1 6 100 97 TN1, TN2, TN8, TN10 Halophytophthora Halophytophthora 127446 29386TN3, TN6 batemanensis Phytopythium sp. 795339 1 99 89 TN10

TABLE II. Summary data for oomycetes from the spring and fall baiting sessions 2010–2012 Genus/species Number of isolates Locations Season Phytophthora species P. cryptogea 17 TN2, TN3,TN4, TN5, TN6, Spring TN7, TN8, TN9 70 TN1, TN2, TN3,TN4, TN5, Fall TN6, TN7, TN8, TN9, TN10, TN11, TN12,TN13, TN15, TN16 P. hydropathica 7 TN1, TN2, TN4,TN7 Spring 14 TN1, TN2, Fall TN4,TN7,TN10,TN12 P. irrigata 2 TN3, TN8 Spring 5 TN2, TN3,TN4, TN7, TN9 Fall P. gonapodyides 1TN9 Spring 4 TN1, TN2 Fall P. “Pgchlamydo” 5 TN7, TN10,TN13 Fall P. lacustris 4 TN2, TN6 Fall P. polonica 1TN6 Spring 2 TN7 Fall Phy Unk1 2 TN2, TN9 Spring 5 TN1,TN7, TN9 Fall Phy Unk2 3TN5 Spring Phy Unk3 1 TN1 Fall Phy Unk4 1 TN7 Fall P. citricola complex 5 TN2, TN3, TN6, TN9 Spring 2 TN10 Fall

Pythium species P. citrinum 1TN5 Spring 6 TN1, TN2, TN3, TN15 Fall P. diclinum 5 TN3, TN7 Spring TN1, TN2, TN3, TN5, Fall 19 TN6, TN7, TN13 P. helicoides 1TN1 Spring 1 TN4 Fall P. litorale 39 TN1, TN2, TN3, TN4, Spring TN5, TN6, TN7, TN8, TN9, TN10 73 TN1, TN2, TN3, TN4, Fall TN5, TN6, TN7, TN8, TN11, TN12, TN13, TN14, TN15, TN16 P. senticosum 1TN5 Spring P. undulatum 3 TN2, TN4 Spring 1 TN5 Fall P. vexans 1TN7 Spring P. apleroticum 1 TN3 Fall P. chamaihyphon 1 TN11 Fall P. montanum 1 TN13 Fall P. pyrilobum 2 TN1 Fall Py Unk1 11 TN1, TN3, TN5, TN6, Spring TN9, TN10 23 TN2, TN3, TN4, TN6, Fall TN7, TN8, TN9, TN10 Py Unk2 1TN9 Spring 3 TN4, TN7, TN13 Fall Py Unk3 5 TN1, TN2, TN8, TN10 Spring 1 TN8 Fall Halophytophthora Halophytophthora batemanensis 2 TN3, TN6 Fall Phytopythium sp. 1 TN10 Spring