Mycol. Res. 108 (7): 775–780 (July 2004). f The British Mycological Society 775 DOI: 10.1017/S0953756204000541 Printed in the United Kingdom.

Plectosporium alismatis comb. nov. a new placement for the pathogen Rhynchosporium alismatis

Wayne M. PITT1*, Stephen B. GOODWIN2, Gavin J. ASH1, Norma J. COTHER3 and Eric J. COTHER3 1 Farrer Centre, School of Agriculture, Charles Sturt University, P.O. Box 588, Wagga Wagga, New South 2678, . 2 Crop Production and Pest Control Research Unit, USDA Agricultural Research Service, Department of Botany and Pathology, 915 West State Street, Purdue University, West Lafayette, IN 47907-2054, USA. 3 New South Wales Agriculture, Agricultural Research Institute, Forest Road, Orange, New South Wales 2800, Australia. E-mail : [email protected]

Received 21 July 2003; accepted 25 April 2004.

The phytopathogen Rhynchosporium alismatis, occurring on , Sagittaria and other genera in the Alismataceae, is under investigation as a mycoherbicide for alismataceous weeds in Australian rice crops. The type species of Rhynchosporium, R. secalis, belongs in the Helotiales while the phylogenetic relationships of R. alismatis were unknown. To identify fungi related to R. alismatis, the internal transcribed spacer (ITS) region of the ribosomal DNA (ITS1, 5.8S rRNA gene, ITS2) of 56 isolates was sequenced and compared to those available in databases. Analysis of ITS sequences revealed close relationships between R. alismatis and the teleomorph genus Plectosphaerella, as well as several anamorphic fungi which were primarily species of Verticillium. Rhynchosporium alismatis and Plectosphaerella cucumerina clustered together with 98% bootstrap support. Morphological comparisons supported this relationship indicating that R. alismatis and the anamorphic genus Plectosporium are congeneric. Rhynchosporium alismatis is transferred to Plectosporium, a name proposed for conservation.

INTRODUCTION for biological control of alismataceous weeds in Australia (Cother & Gilbert 1994a, b). Rice cultivation in Australia is confined to the irrigated The genus Rhynchosporium was described by Heinsen regions of southern New South Wales and northern (1901), based on the type species R. graminicola (i.e. Victoria. Several members of the Alismataceae, includ- R. secalis), the cause of barley scald. At present only ing Alisma lanceolatum, A. plantago-aquatica and two species are recognised (Braun 1995): R. secalis, and Damasonium minus are important aquatic weeds of rice R. orthosporum which produces similar disease symp- crops in these areas (McIntyre et al. 1991). At present, toms on orchardgrass (Dactylis glomerata) (Caldwell control options for these weed species are limited and 1937). The name R. alismatis was originally proposed depend almost entirely on application of chemical as a new combination for Septoria alismatis by Davis herbicides. (1922). The primary morphological difference noted by Cother, Gilbert & Pollock (1994) reported the effects Davis was the lack of any apical bending of the conidia of the naturally occurring fungus, Rhynchosporium of R. alismatis compared to those of R. secalis. How- alismatis, on the growth of A. lanceolatum and later ever, Caldwell (1937) examined fresh material supplied demonstrated its pathogenicity to, and growth sup- by Davis and concluded that it was different from the pression of, five other Alismataceae species including other species in the genus in the lack of a superficial A. plantago-aquatica, D. minus and three species of fertile stroma and production of conidia on short Sagittaria (Cother & Gilbert 1994a). Due to its sup- flask-shaped conidiophores. Therefore, Caldwell (1937) pression of plant growth and host specificity, R. alis- excluded R. alismatis from Rhynchosporium, but did not matis has been proposed as a potential mycoherbicide rename it or even speculate on its possible relation- ships. This uncertain status remained until recently * Present address: Agriculture and Agri-Food Canada, Saskatoon Research Centre, 107 Science Place, Saskatoon, SKS7N 0X2, when R. alismatis was transferred to the new genus Canada. Spermosporina (Braun 1993). Rhynchosporium alismatis congeneric with Plectosporium 776

Despite this taxonomic revision, current publications for 48 h and ground in liquid nitrogen for DNA continue to refer to the organism as R. alismatis (e.g. extraction. Cother & van de Ven 1999, Fox, Cother & Ash 1999, Lanoiselet et al. 2001, Jahromi, Cother & Ash 2002), DNA extraction, amplification and sequencing and nothing is known about its phylogenetic relation- ships. Braun (1993) suggested that his new genus For DNA extraction, a 2 ml microcentrifuge tube was Spermosporina was closely related to Rhynchosporium. filled to the top of the conical portion (approx. 0.5 ml) Recent phylogenetic analyses have revealed that R. with ground, lyophilised mycelium and 800 ml of lysis secalis and R. orthosporum, are closely related to the buffer (1% sodium dodecyl sulphate, 10 mM Tris-HCl discomycete genera Tapesia and Pyrenopeziza in the pH 8.0, 0.5 M NaCl and 10 mM EDTA) was added. The Helotiales (Goodwin 2002), but those analyses did not extraction was then performed according to the method include R. alismatis. If Braun’s (1993) hypothesis is of Sambrook, Fritsch & Maniatis (1989). DNA was correct, R. alismatis also should be related to Helotiales. visualized by electrophoresis on 1% agarose containing Because regulatory agencies require definitive taxo- 1rTris acetate-EDTA (TAE; Sambrook et al. 1989) nomic knowledge of fungi intended for release as bio- stained with 0.3 mgmlx1 ethidium bromide. DNA was control agents and information on their relationships adjusted to 10 ng mlx1 via the addition of TE buffer with known crop pathogens, this may affect develop- (10 mM Tris-HCl pH 8.0 and 0.1 mM EDTA) and ment options; the phylogenetic affinities of the fungus stored at x20 x. therefore must be determined. The complete ITS region (ITS1, 5.8S, ITS2) of each The purpose of this study was to determine the isolate was amplified with primers AB28 and TW81 of affinities of R. alismatis to other fungi using the ITS Howlett et al. (1992). PCR reactions were performed in region of rDNA (ITS1, 5.8S, and ITS2). This region a total volume of 50 ml by mixing 35 ng of purified represents that for which the largest number of se- genomic DNA with 100 pmol each of primers AB28 quences is available in public databases. Hence, com- and TW81, 0.1 mM each of dATP, dCTP, dGTP and parisons of this gene region provide the greatest dTTP (Amersham Pharmacia Biotech, Castle Hill, likelihood of identifying close relatives for this species. Australia), 2 mM MgCl2 and 2 units of Taq DNA polymerase (Promega Corporation, Annandale, Australia) in PCR reaction buffer (50 mM Tris-HCl MATERIALS AND METHODS pH 8.0, 100 mM NaCl, 0.1 mM EDTA, 1 mM DTT, 50% glycerol and 1% Triton X-100). The reaction was Sources and culturing of isolates performed with an initial denaturation of 5 min at 56 isolates of Rhynchosporium alismatis were obtained 95 x followed by 35 cycles of denaturation (45 s at 94 x), from several locations throughout Australia, Asia annealing (30 s at 55 x) and extension (90 s at 72 x), and and during the period Jan. 1989–Sept. 2003. a final extension (5 min at 72 x). PCR was performed in Isolates obtained from Australia and Asia were a Corbett Research FTS 960 DNA thermal cycler purified from infected of host , cultured on (Corbett Research, Mortlake, Australia). PCR pro- quarter-strength potato dextrose agar (PDA; Oxoid ducts were visualised by electrophoresis as described Australia, West Heidelberg, Australia) supplemented previously. All PCR amplifications were performed in with 10 mg lx1 rifampicin, 250 mg lx1 ampicillin and duplicate to confirm that results were reproducible. 100 mg lx1 pentachloronitrobenzene (Sigma-Aldrich, Following electrophoresis, ITS products of all 56 Castle Hill, Australia), lyophilised and deposited in the isolates were purified with the Wizard PCR Preps DNA Agricultural Scientific Collections Unit, New South Purification System (Promega Corporation). Sequen- Wales Agriculture, Orange, NSW, Australia (DAR). cing of the purified PCR products was performed by Isolates from Europe were obtained as freeze-dried Newcastle DNA Express Sequencing (Newcastle, cultures from Walter Gams, (CBS, Utrecht) and re- Australia). PCR products were sequenced in an ABI vived by E.J.C. (DAR). PRISM 377 DNA thermocycle sequencer (Perkin Prior to experimentation, all isolates were single- Elmer Applied Biosystems, Sydney) using fluorescent spored on PDA, before being transferred to lima bean dideoxy (dye-labelled)-nucleoside-triphosphate termin- agar (LBA; Difco Laboratories, Detroit, MI) and in- ators. Sequence mismatches were corrected visually cubated for 5–10 d at 25 xC. Spores were harvested using the ABI PRISM electropherograms in conjunc- from each culture by adding 1 ml of sterile distilled tion with the computer package Chromas 1.56 (Tech- water and scraping gently across the mycelium surface nelysium, Helensvale, Australia). with a sterile glass rod. The resulting slurry of conidia and mycelium was used to inoculate 250 ml Erlenmeyer Assembling the ITS database flasks containing 150 ml of clarified 20% (v/v) V8 juice (Campbell’s Soup, Lemnos, Australia). Flasks To identify species that may be closely related to were then incubated on an orbital shaker at 100 rpm Rhynchosporium alismatis, a BLAST (Altschul et al. for up to 14 d at 25 x. When sufficient growth had oc- 1997) search was performed using the ITS sequence curred, mycelia were harvested by filtration, lyophilised from R. alismatis isolate RH01. Sequences with high W. M. Pitt and others 777

Table 1. Summary information for isolates included in the ITS Statistical support for inferred groups was estimated a sequence database . by bootstrap analysis (Felsenstein 1985) using 1000 GenBank replications and was performed using the Bootstrap accession NJ-tree option. The final tree was displayed with Species Isolate no. NJplot (Perriere & Gouy 1996).

Plectosphaerella cucumerina NRRL 20430 AF176952 P. cucumerina 00017 AJ246154 P. cucumerina 380408 AJ492873 RESULTS P. cucumerina RH126 AY258149 Rhynchosporium alismatisb RH01 AY258151 PCR with the oligonucleotide primers AB28 and TW81 b R. alismatis RH62 AY258150 amplified a single DNA fragment that migrated be- R. alismatisb Gams2A AY572015 R. alismatisb Gams1B AY572020 tween the 501 and 710 bp length markers for each of Verticillium albo-atrum UAMH 5393 AF108476 the isolates sequenced in this study. Following se- V. albo-atrum KRS1 AF364008 quence analysis and subsequent removal of terminal V. albo-atrum 166 AF364016 primer sequences, the length of the ITS region ampli- V. albo-atrum – L19499 fied from each of the isolates was 501 bp. This stretch V. albo-atrum ATCC 44943 X60705 V. albo-atrum 1776 Z29509 included the entire ITS region (ITS1, 5.8S, ITS2), 13 bp V. albo-atrum 2 Z29523 of the 18S ribosomal DNA subunit and 24 bp of the V. dahliae UAMH 5360 AF108478 large ribosomal DNA subunit. The Rhynchosporium V. dahliae 001 AF363986 alismatis ITS region alone spanned 464 bases: 136 V. dahliae MD80 AF364004 bases for ITS1, 159 for the 5.8S region and 169 V. dahliae 2341 Z29511 V. nigrescens UAMH 6687 AF108473 for ITS2. Intraspecific sequence variation between the V. nigrescens IMI 044575 AJ292440 R. alismatis sequences was minimal, with 53 of the V. nubilum IMI 130213 AJ292463 isolates identical for this region. Three isolates, RH62, V. tricorpus 1988 AF364017 Gams2A and Gams1B exhibited slight variation. RH62 V. tricorpus – L28679 contained two transition mutations within the ITS1 V. tricorpus 267 Z29524 region at positions 71 (T to C) and 125 (C to T), a according to Kirk et al. (2001). Gams2A contained one transition and one transversion b Duplicate cultures held at DAR and/or CBS for RH01 (DAR mutation within ITS2 at positions 472 (A to G) and 475 67515, CBS 112531), RH62 (DAR 67513, CBS 112535), Gams2A (DAR 76106, CBS 113362) and Gams1B (DAR 76496). (A to C) respectively, and Gams1B contained a single transversion mutation within the ITS1 region at position 15 (T to G). Isolates RH62, Gams2A, Gams1B similarity to RH01 were downloaded from GenBank and RH01 were chosen for inclusion in the final ITS and used to compile an ITS database. Multiple se- database, with the latter isolate representing the 53 quences of the same species were removed for the final identical sequences. Sequence data for all 56 isolates analysis unless their sequences differed. The final data- analysed in this study were submitted to the GenBank base contained sequences from 25 isolates comprising database. Isolates included in the final ITS database seven species from three genera (Table 1). were designated accession nos. AY258150, AY572015, AY572020 and AY258151, respectively. A BLAST search of the GenBank database using Sequence alignment and analysis R. alismatis RH01 identified strong matches with many All sequences, including those downloaded from Gen- species of Verticillium and Trichoderma, as well as the Bank, were trimmed to include the complete ITS region teleomorph genera Nectria, Neonectria,andHypocrea. (ITS1, 5.8S, and ITS2). Where available, seven bases The highest BLAST scores were obtained to isolates of each of the 18S and large ribosomal RNA subunit were Plectosphaerella cucumerina, followed by Verticillium retained to aid in the alignment. Individual sequences nigrescens and V. nubilum. Based on these results, were compiled in BioEdit Sequence Alignment Editor 21 sequences representing six species and two genera (Hall 1999) and analysed by ClustalX (Thompson et al. were downloaded from GenBank and used to compile 1997). A multiple sequence alignment comprising all 25 an ITS database. With the addition of ITS sequences sequences was performed and the Draw NJ-tree option from the four R. alismatis isolates and one new isolate was utilised to produce a neighbour-joining tree of the of P. cucumerina (RH126) generated de novo, the final relationships between the isolates. The Draw NJ-tree database contained 25 sequences comprising seven function calculates genetic distances among isolates species from two anamorphic and one teleomorphic using Kimura’s (1980) two-parameter method for esti- genera (Table 1). mating evolutionary distances and prepares a phylo- To study the relationships between isolates in the ITS genetic tree based on the neighbour-joining algorithm database, a phylogenetic tree was produced according of Saitou & Nei (1987), equivalent to the DNADIST to the neighbour-joining method (Saitou & Nei 1987). and NEIGHBOR programs of the PHYLIP pack- Confidence values for the inferred groups were esti- age (Felsenstein 1989, Goodwin & Zismann 2001). mated by bootstrap analysis (Felsenstein 1985). The ITS Rhynchosporium alismatis congeneric with Plectosporium 778

0.02 Verticillium albo-atrum- 2 71 Verticillium albo-atrum- Verticillium albo-atrum 166 77 Verticillium albo-atrum ATCC 44943 Verticillium albo-atrum- UAMH 5393 Verticillium albo-atrum- KRS1 Verticillium dahliae 001 72 Verticillium albo-atrum- 1776 Verticillium dahliae MD80 86 94 Verticillium dahliae UAMH 5360 Verticillium dahliae 2341 Verticillium nubilum Plectosphaerella cucumerina NRRL 20430 98 85 Plectosphaerella cucumerina 380408 Plectosphaerella cucumerinaRH126 98 85 95 Plectosphaerella cucumerina 00017 Rhynchosporium alismatisGams2A 89 97 Rhynchosporium alismatis RH62 100 Rhynchosporium alismatis Gams1B Rhynchosporium alismatis RH01 Verticillium nigrescens UAMH 6687 100 100 Verticillium nigrescens IMI 044575 Verticillium tricorpus 267 Verticillium tricorpus 1988 76 Verticillium tricorpus

Fig. 1. Neighbour-joining tree of ribosomal DNA ITS sequences from Rhynchosporium alismatis and related species. Bootstrap values of 70 or greater (percentage of 1000 replications) are indicated, rounded to the nearest integer. If more than one isolate of a species was analysed, isolate designations are provided after the species name. Branch lengths are proportional to genetic distance, which is indicated by a bar at the upper right. alignment showed that R. alismatis is closely related ochraeceus to apricot (more intense in R. alismatis on to P. cucumerina, V. nigrescens and several other synthetic nutrient-poor agar (SNA) medium than in P. species of Verticillium (Fig. 1). Sequences for isolates of tabacinum), and with a felty or woolly appearance and P. cucumerina and R. alismatis grouped together with often with radiating concentric rings. Microscopically, 98% bootstrap support. Isolates of R. alismatis formed conidia of both species are mostly ellipsoid, almost a monophyletic group basal to Plectosphaerella with straight to slightly curved, often appearing solitary on 97% bootstrap support. the tip of the conidiogenous cell or in groups, smooth, hyaline, tapering to a broadly rounded apex and to a narrower truncate base, and multiguttulate when DISCUSSION mounted in water. Conidiogenous cells are hyaline, The analysis of ITS sequences reveals that Rhynchos- smooth, discretely phialidic with a short collarette in porium alismatis is clearly not related to the generic both species and arise on the host from hyphal ag- type species R. secalis, nor its close relative R. orthos- gregations. Additionally, both species have similar porum, in the Helotiales. Instead, R. alismatis proves nutritional and environmental requirements during to be congeneric with Plectosporium tabacinum in growth and sporulation (Jahromi, Ash & Cother 1998, the Phyllachorales. This fungus is connected with a Zhang, Sulz & Bailey 2001), produce similar disease teleomorph in Plectosphaerella, which is related to symptoms on susceptible hosts and have overlapping Glomerella, commonly classified in the Phyllachorales host ranges that include members of the Alismataceae, (Silva-Hanlin & Hanlin 1998) but representing a dif- specifically Sagittaria pygmaea (Cother & Gilbert ferent order (Winka & Eriksson 2000). Hence, Braun’s 1994a, Chung et al. 1998), Cucurbitaceae and Solana- (1993) conclusion that the genus Spermosporina is dis- ceae, from which R. alismatis was isolated on several tinct and separate from Rhynchosporium is correct for occasions following host-range studies (Cother 1999). R. alismatis, although the two genera are evidently Plants from the latter two families were also identified unrelated. as potential hosts by Zhang et al. (2002). However, in R. alismatis and P. tabacinum show many simi- both cases P. tabacinum was shown to cause only a low- larities. In culture both fungi are similar, with colony grade non-progressive disease on cucurbits and tom- pigmentation ranging from off-white through pale ato, similar to that incited by R. alismatis. Similarly, W. M. Pitt and others 779

P. tabacinum caused only partial blight of jimson Rhynchosporium alismatis (Oudem.) Davis, Trans. Wis. Acad. weed (Datura stramonium) when tested by Chung et al. Sci. Arts Lett. 20: 420 (1922). (1998), suggesting that both R. alismatis and P. taba- Spermosporina alismatis (Oudem.) U. Braun, Cryptog. Bot. 4: cinum are only weak pathogens of cucurbits and sola- 111 (1993). naceous hosts. Some differences are also apparent. Conidia of R. Ramularia sagittariae Bres., Hedwigia 36: 200 (1896). alismatis are mainly 1-septate whilst those of P. taba- Spermosporina sagittariae (Bres.) U. Braun, Cryptog. Bot. 4: cinum are often non-septate or less than 50% are sep- 113 (1993). tate (Palm, Gams & Nirenberg 1995). When mounted in lactic acid, conidia of R. alismatis are more slender, Type:OnAlisma plantago-aquatica, described by Pu- consistently banana-shaped and chlamydospores are nithalingam (1988). To fix the molecular identity of the spe- cies, we designate an epitype: The Netherlands: Pijnenburg nr regularly present (Walter Gams, pers. comm.). Fur- Soest, ex- spots on Alisma plantago-aquatica, July 2003, thermore, conidia in P. tabacinum are produced from W. Gams (CBS 113362 – epitypus hic designatus). The epitype unbranched or infrequently branched conidiophores, is available as living ex-epitype cultures as well as the desig- as opposed to the short flask-shaped conidiophores nated dried reference specimen. typical of R. alismatis (Caldwell 1937), which combined with a lack of superficial stroma was evidence enough for this author to exclude it from Rhynchosporium. Additionally, conidiogenesis in R. secalis and R. ACKNOWLEDGEMENTS orthosporum is blastic (Punithalingam 1988), another This work was supported by the Cooperative Research Centre for major distinction from R. alismatis. Sustainable Rice Production project 2401, financial assistance pro- Despite the morphological, developmental and vided by the Charles Sturt University Write-Up and Special stipend epidemiological similarities between R. alismatis, P. Schemes and by USDA CRIS project 3602-22000-011-00D. We wish to thank Uwe Braun and Mary Palm for helpful discussions, and tabacinum and its teleomorph P. cucumerina, the cor- Walter Gams for critical evaluation of the manuscript. rect classification of R. alismatis has been problematic. For instance, the Plectosphaerella/R. alismatis cluster is embedded between V. nigrescens and several other species of this genus as described previously by Zare, REFERENCES Gams & Culham (2000). However, Plectosporium is morphologically distinct from Verticillium and Altschul, S. F., Madden, T. L., Schaffer, A. A., Zhang, J., Zhang, Z., V. nigrescens is the least characteristic species of this Miller, W. & Lipman, D. J. 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