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A new species of Pyricularia (hyphomycetes) on Cortaderia () in New Zealand

McKenzie EHC*, Park D, Bellgard SE and Johnston PR

Manaaki Whenua Landcare Research, Private Bag 92170, Auckland, New Zealand [email protected] [email protected] [email protected] [email protected]

McKenzie EHC, Park D, Bellgard SE, Johnston PR. 2010 – A new species of Pyricularia (hyphomycetes) on Cortaderia (Poaceae) in New Zealand. Mycosphere 1(3), 223–228.

Pyricularia cortaderiae sp. nov., found on leaves of in New Zealand, is illustrated and described and compared with related taxa. rDNA sequencing showed it to be distinct from other species. It was associated with a narrow, dark brown leaf streak.

Key words – anamorphic fungi – deuteromycetes – molecular phylogeny –

Article Information Received 22 September 2010 Accepted 28 September 2010 Published online 30 October 2010 *Corresponding author: Eric McKenzie – e-mail – [email protected]

Introduction structures were removed, mounted in lacto- Cortaderia species are tall, perennial, phenol, and examined by light microscopy. tussock grasses found mainly in South America. Measurements were made on material mounted However, there are five endemic species in lactophenol. Single conidia of the (toetoe) in New Zealand and two species (pam- were removed from the tissue and grown pas grass) naturalized from South America. on potato dextrose agar (PDA). A living culture The naturalized species commonly grow in is preserved in the International Collection of waste places and scrub and invade Pinus Micro-organisms from (ICMP). A dried plantations. While carrying out a survey of herbarium specimen of the fungus was pre- pampas grasses for possible biological control pared and is deposited in the New Zealand agents, a species of Pyricularia was found. It is Fungal Herbarium (Herb. PDD). The species is described below as a new species. known only from the holotype. Fungal DNA was extracted from Methods mycelium of P. cortaderiae grown on a PDA Leaves and stems of a naturalized, wide- plate using REDExtract-N-Amp Plant PCR spread pampas grass, Cortaderia selloana Kits (Sigma, USA), following the manufac- (Schult. & Schult. f.) Asch. & Graebn. were turer’s instructions. The ITS region was collected from a scenic reserve. The Corta- amplified using the primers ITS1F and ITS4 deria was growing as a large clump within a (White et al. 1990). The newly obtained ITS patch of the endemic sedge, Gahnia xantho- sequence was aligned using Clustal W (Larkin carpa. The plant material was incubated under et al. 2007) with sequences available from humid conditions and periodically examined Bussaban et al. (2005) that represented the for sporulating microfungi. Fungal fruiting genetic breadth of the Pyricularia spp. sampled

223 in that study, along with ITS sequences from Cannaceae, Commelinaceae, Cyperaceae, Mu- other grass-inhabiting Pyricularia spp. availa- saceae, and Zingiberaceae. The species on ble in GenBank. Phylogenetic analyses were monocotyledonous plants usually cause leaf performed using Bayesian maximum likleli- spots, but some other species are saprobic on hood in MrBayes 3.1.2 (Huelsenbeck & leaf litter of dicotyledonous plants. Ronquist 2001) and a heuristic maximum The is characterised by the forma- likelihood analysis in PAUP* 4.01b (Swofford tion of solitary conidia on cylindrical denticles 2002) with the GTR+I+G model, selected produced from usually slender, thin-walled using the AIC method in MrModelTest 2.3 conidiophores. The conidiogenous cells are (Posada & Crandall 1998, Posada & Buckley terminal, sometimes becoming intercalary 2004). The Bayesian analysis was run with two through sympodial extension of the conidio- chains for 10 million generations, trees genous cell and formation of new cross walls. sampled every 1000 generations with a burn-in The conidia are often 1- or 2-septate, and of 25%. Bayesian posterior probabilities were obpyriform, obturbinate or obclavate in shape obtained from 50% majority rule consensus (Ellis 1971a). trees. The PAUP ML analysis used addition A specimen collected on Cortaderia sequence random and TBR branch swapping selloana in New Zealand is distinct from all with 100 replicates to avoid local optima. A other known species, and is described as: bootstrap analysis used the ML tree as a starting tree, each of the 100 bootstrap samples Pyricularia cortaderiae McKenzie, sp. nov. run with a single replicate. Fig. 1 MycoBank 512735 Results GenBank accession number: HQ283076 The genus Pyricularia Sacc. was des- Etymology – named after the host cribed by Saccardo (1880) for a single species, substrate, Cortaderia. P. grisea Sacc., found on grasses. A second Coloniae pilosae. Mycelium ex hyphis in species, P. oryzae Cav., was later described on substrato immersum, ramosis, septatis, laevibus, rice (Cavara 1892). Various subspecific taxa hyalinae vel luteus vel pallide brunneis, tenuitu- have been described within both of these nicatis, 1.5–3 µm crassis compositum. Conidio- species, often based on differences in host phora macronematosa, mononematosa, solitaria, pathogenicity. While these two species are erecta, recta vel paulo flexuosa, nonramosa, morphologically similar, recent pathogenicity laevia, 2–5-septata, pallide brunnea vel brunnea, and mating studies (Kato et al. 2000), mole- apicem versus pallidiora, usque ad 250 µm cular studies (Couch & Kohn 2002) and des- longa, 5.5–7 µm lata. Cellulae conidiogenae criptions of teleomorphs ( holoblasticae, in conidiophora incorporatae, (T.T. Hebert) M.E. Barrand and M. oryzae B.C. terminales, sympodiales, cylindricae, usque ad Couch), have led to the conclusion that these 155 µm longa, denticulis conico-truncatis vel two species are distinct with P. oryzae parasitic cylindricis numerosis praedita. Conidia solitaria on rice and many grasses, and P. grisea ex denticulis oriunda, sicca, acropleurogena, parasitic on spp. (Couch & Kohn straminea vel pallide brunnea, laevia, obovoidea 2002). A molecular study by Hirata et al. (2007) vel obpyriformea, basi truncate, apice late revealed cryptic species within the P. grisea rotundato, 2-septatis, 25.5–32 × 11.5–15 µm. complex. Other species have been described on Type: NEW ZEALAND, Auckland, grasses: P. didyma M.B. Ellis, P. dubiosa Pakiri, Pakiri Scenic Reserve, 36° 12.44830’S, (Speg.) Viégas, P. leersiae (Sawada) S. Ito, P. 174°39.03433’E, in foliis vivis poacearum panici-paludosi (Sawada) S. Ito, P. penniseti Cortaderia selloana (Poaceae), 17 November Prasada & Goyal, P. setariae Y. Nisik, and P. 2008, R.E. Beever, S.E. Bellgard, N.W. zizaniicola Hashioka. In addition, several Waipara (PDD 95061, holotype) (culture from invalid species have been named for Pyricula- type specimen, ICMP 17830). ria on grasses (Siwasin & Giatgong 1971). Colonies in the form of scattered conidio- Additional species have been described, espe- phores. Mycelium immersed in the substratum. cially on monocotyledonous plants including Hyphae branched, septate, smooth, hyaline to

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Fig. 1 – Conidia and conidiogenous cells of Pyricularia cortaderiae (from holotype). Specimen mounted in hydrous lactophenol. Scale bar = 20 µm. yellowish, thin-walled, 1.5–3 µm diam. Coni- Fig. 2. The relationship of P. cortaderiae was diophores differentiated, mononematous, single, unresolved within Pyricularia. erect, straight or slightly flexuous, unbranched, smooth, 2–5-septate, pale brown to brown, Discussion sometimes paler towards apex, up to 250 µm The species of Pyricularia that cause leaf long, 5.5–7 µm wide. Conidiogenous cells spots on grasses and cereals are morpholo- holoblastic, integrated, terminal, sympodially gically similar to P. cortaderiae, possessing proliferating, cylindrical, up to 155 µm long, conidia that are pale coloured, 2-septate (except with conspicuous denticles; denticles conico- for P. didyma, which is 1-septate), and obovoid, truncate, up to 3 µm high, 3 µm wide. Conidia obpyriform or obturbinate in shape (Table 1). solitary at ends of denticles, dry, acropleuro- However, the conidia of P. cortaderiae are genous, straw-coloured or pale brown, smooth, larger than those of all of the other species, obovoid to obpyriform, base truncate, apex except for P. zizaniicola. The conidial size broadly rounded, 2-septate, 25.5–32 × 11.5–15 range and mean dimensions of P. cortaderiae µm (mean = 29.3 × 13.7 µm, n = 30). and P. zizaniicola are virtually identical (Table Pyricularia cortaderiae was found 1). The main morphological difference between sporulating exclusively on old, narrow, elon- these two species appears to be in the conidio- gate, sometimes coalescing, dark brown leaf phores. The conidiophores of P. zizaniicola are streaks (1–2 mm wide × 1–2 cm long) on predominantly 0–1-septate, rarely 2-septate, Cortaderia selloana, and is presumably a and measure 90–150 µm, rarely extending to pathogen. 220 µm (Hashioka 1973). Those of P. cortaderiae are 2–5-septate and up to 250 µm Phylogenetic analyses in length. Both the ML and Bayseian analyses of CAB International (2002) gave conidial ITS rDNA sequences produced a tree with the measurements of 19–31 × 10–15 µm for P. same topology. The Bayesian tree in shown in setariae. This is somewhat larger than the 225 Fig. 2 – 50% majority-rule consensus phylogenetic tree based on Bayesian analysis of the ITS region. Bayesian posterior probabilities/ML bootstrap values are shown above the edges where they are greater than 50%. The sequence for Pyricularia cortaderiae newly generated in this study has been deposited in GenBank as HQ283076. Nakatea fusispora and Dactylaria lanosa were selected as outgroups to root the tree.

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Table 1 Summary of features of Pyricularia species found on grasses.

Species Conidia Conidiophores Size (µm) Mean (µm) Septa Size (µm) P. cortaderiae (current 25.5–32 × 11.5–15 29.3 × 13.7 2 Up to 250 × 5.5–7 material) P. didyma (Ellis 1971b) 12–16 × 7.5–8.5 14 × 8 1 Up to 200 × 2.5–4.5 P. dubiosa (Viégas 1946) 15–22 × 8–10 – 2 80–100 × 3.5–4 P. grisea (Ellis 1971a) 17–28 × 6–9 20.9 × 7.7 2 Up to 150 × 2.5–4.5 P. leersiae (Tanaka 1920) 20–35 × 7–10 27 × 8.6 2 48–88 × 4–5 P. oryzae (Ellis 1971a) 17–23 × 8–11 21.2 × 9.6 2 Up to 130 × 3–4 P. panici-paludosi (Tanaka 17–26 × 8.5–12 22 × 10.2 2 80–160 × 4–5 1920) P. penniseti (Prasada & Goyal 18.4–36.7 × 7.4–11.1 27.5 × 9.2 2 50–150 × 3.6–5.5 1970) P. setariae (Nishikado 1917) 14–35 × 5–12 20 × 7.5 (1–)2(–3) 40–250 × 4-5 P. zizaniicola (Hashioka 1973) 24–33 × 10.5–15.5 27.7 × 13.5 2 90–150(–220) × 7–9 measurements given by Nishikado (1917) and in New Zealand it is common on dead leaves of places the size limits within the range of P. Cyperaceae (Uncinia and Carex species). cortaderiae. Hirata et al. (2007) designated a The phylogenetic relationships within cryptic, phylogenetic species as Pyricularia sp., Pyricularia sens. lat. are poorly resolved, within the P. grisea complex. It was isolated probably reflecting the limited gene sampling from both geniculata and Leersia in studies on the genus to date (Bussaban et al. oryzoides. Following DNA analysis, Yamaga- 2005, Hirata et al. 2007). The position of P. shira et al. (2008) designated Pyricularia cortaderiae is unresolved, although appears to isolates from both wild foxtails (Setaria spp.) be far removed from other grass-inhabiting and foxtail millet (S. italica) as the Setaria species, and somewhat related to lineages of pathotype of Magnaporthe oryzae. and to some of the Pyricula- Sequencing of the ITS-region showed ria spp. occurring on gingers. that P. cortaderiae is distinct from any of the other species recorded on grasses, for which Acknowledgements sequences are available (Fig. 2). It is distinct Funds for this research were provided by from P. zizaniicola and isolates originating the New Zealand Foundation for Research, from Setaria italica and S. geniculata (Fig. 2). Science and Technology through the Defining It is worth noting that the genus Cortaderia lies New Zealand’s Land Biota OBI. Paul Cannon, within subfamily Arundinoideae of the Poaceae CABI, UK is thanked for kindly providing while Zizania (host of P. zizaniicola) is in comments on the manuscript. subfamily Bambusoideae, and Setaria (host of P. setariae and an unnamed Pyricularia sp. References (Hirata et al. 2007)) is in subfamily Panicoi- deae (Clayton & Renvoize 1986). Pyricularia Bussaban B, Lumyong S, Lumyong P, cortaderiae lies within a lineage that includes Seelanan T, Park DC, McKenzie EHC, P. juncicola MacGarvie. However, it is mor- Hyde KD. 2005 – Molecular and phologically distinct as conidia of the latter are morphological characterization of Pyri- 1-septate, obclavate, and measure 15–30 × 4–5 cularia and allied genera. Mycologia 97, µm (Ellis 1976). P. juncicola was described 1002–1011. from Ireland on dead leaves of Juncus effusus;

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