Four new species in Magnaporthaceae fromgrass roots in New Jersey Pine Barrens

Jing LuoEmily WalshNing Zhang1

Department of Plant Biology and Pathology, 201 ForanHall, 59 Dudley Road, Rutgers University, NewBrunswick, New Jersey 08901

Abstract: Based on morphology and DNA sequencesof SSU, ITS, LSU, MCM7, RPB1 and TEF1 genes, wedescribe four new species in Magnaporthaceae thatare associated with grass roots collected from NewJersey Pine Barrens. A new genus, Pseudophialophora,is erected to accommodate three species, which ischaracterized by slow growth on potato dextrose agar,curved conidiogenous cells without a conspicuouscollarette at the apex and oblong ellipsoidal conidia.Pseudophialophora eragrostis, P. panicorum and P.schizachyrii are assigned to this genus. A new speciesof Magnaporthiopsis also is reported and named as M.panicorum. Distinctions between them and phyloge-netic relationships with other Magnaporthaceae taxaare discussed.

Key words: Harpophora, Magnaporthe, multigenephylogeny, Phialophora, Pseudophialophora, Pyricu-laria, systematics, taxonomy

INTRODUCTION

Phialophora Medlar is a hyphomycetous genus andtypified by Phialophora verrucosa Medlar (Medlar1915). The main characteristics of the genus aredarkly pigmented hyphae, simple or branched conid-iophores and phialidic conidiogenous cells with aflaring collarette at the apex (Cain 1952, Barnett andHunter 2006). It is highly polyphyletic and found tobe connected to a number of teleomorphic taxa invarious orders, such as Caliciales, Chaetothyriales,Diaporthales, Dothideales, Hypocreales, Leotiales,Magnaporthales, Ophiostomatales, Sordariales andSpathulosporales (Gams 2000). Gams (2000) intro-duced a new asexual genus, Harpophora W. Gams, toaccommodate four Phialophora-like species character-ized by fast growing colonies, pigmented phialideswith a typical collarette, and strongly curved conidiathat were reluctant to germinate on standard media.In Magnaporthaceae, Harpophora-like state was con-

nected to, Buergenerula Syd., Ceratosphaeria Niessland Gaeumannomyces Arx & D.L. Olivier, whilePhialophora-like state was found in MagnaporthiopsisJ. Luo & N. Zhang (Cannon 1994, Reblova 2006,Huhndorf et al. 2008, Zhang et al. 2011, Luo andZhang 2013).

In this study 10 Phialophora-like fungal isolates werefound during our recent survey of fungi associatedwith grass roots in New Jersey Pine Barrens. Based onmorphology, biology, ecology and multigene phylo-genetic analyses, a new genus and four new speciesare proposed in family Magnaporthaceae.

MATERIALS AND METHODS

Fungal isolation.—Healthy grass roots were sampled fromColliers Mills (N40 04.093, W74 26.598) and AssunpinkLake (N40 12.962, W74 30.527) in the New Jersey PineBarrens in Aug 2012. Grass samples were transported to thelaboratory and processed for fungal isolation within 24 h.The roots were rinsed in tap water to remove soil particleson the surface and cut into ca. 5 mm long fragments. Thesefragments were surface-sterilized with 75% alcohol for5 min, followed by 5 min in 0.6% sodium hypochloriteand two final rinses in sterile distilled water (Zhang et al.2011). The disinfected fragments were placed on maltextract agar (MEA, BD) with 0.07% lactic acid andincubated at room temperature. Fungal cultures wereisolated and purified by subculturing from emergent hyphaltips.

Cultural study.—Cultural characteristics were recordedfrom potato dextrose agar (PDA, BD) and cornmeal agar(CMA, BD), and the color names of colonies followedRidgway’s nomenclature (Ridgway 1912). Microscopicexaminations, measurements and images were taken fromslides of fungi mounted in distilled water. The specimensexamined were deposited in the Rutgers MycologicalHerbarium, New Brunswick, New Jersey (RUTPP), andcultures were deposited in the Centraalbureau voorSchimmelcultures Fungal Biodiversity Centre, the Nether-lands (CBS).

DNA extraction, amplification and sequence analysis.—Theprotocols described by Zhang et al. (2011) and Luo andZhang (2013) were used for DNA extraction, PCR amplifi-cation and sequencing of small subunit (SSU), internaltranscribed spacer (ITS), and large subunit (LSU) ofribosomal RNA genes, DNA replication licensing factor(MCM7), the largest subunit of RNA polymerase II (RPB1)and translation elongation factor 1-a (TEF1) genes. In thisstudy 40 isolates representing 18 species and three varieties,were included in the analyses. All taxon names together

Submitted 24 Sep 2013; accepted for publication 16 Dec 2013.1 Corresponding author. E-mail: zhang@aesop.rutgers.edu

Mycologia, 106(3), 2014, pp. 580–588. DOI: 10.3852/13-306# 2014 by The Mycological Society of America, Lawrence, KS 66044-8897

580

with the isolate numbers, sources, hosts and GenBankaccession numbers are listed (TABLE I).

Sequence alignments were constructed with Clustal X 1.8(Thompson et al. 1997) and BioEdit 7.0.5 (Hall 1999). Six-gene datasets were assembled for phylogenetic analyses.Cryphonectria parasitica was used as outgroup taxon.Maximum likelihood (ML) analysis with the selected modelwas carried out in PAUP* 4.0b10 (Swofford 2002). Startingtrees were obtained by random sequence addition with 100replicates in heuristic search. The branch swappingalgorithm was tree-bisection-reconnection (TBR), and bothsteepest descent and MULTREES options were not in effect.Maximum parsimony (MP) analysis was performed withheuristic search in PAUP* 4.0b10 (Swofford 2002). Allcharacters were given equal weight. Gaps were treated asmissing characters. Starting trees were obtained via randomsequence stepwise addition with 1000 replicates. Thebranch-swapping algorithm was tree-bisection-reconnection(TBR). Steepest descent and MULTREES options were not ineffect. To calculate all branch support values, a bootstrapanalysis was performed with 1000 replicates using heuristicsearches with simple sequence stepwise addition for eachreplicate. Bayesian inference (BI) was conducted with theMarkov chain Monte Carlo method in MrBayes 3.2.1(Ronquist et al. 2012) under the nucleotide substitutionmodel selected by using hierarchical likelihood ratio tests(hLRTs) and Akaike information criterion (AIC) inMrModeltest 2.3 (Nylander 2004). Trees were sampledevery 100 generations from 10 000 000 generations resultingin 100 000 trees. The first 25 000 trees were discarded asburn-in and the remaining 75 000 trees were chosen tocalculate posterior probability values of clades in aconsensus tree.

RESULTS

A total of 504 nucleotide characters including gapswere in the SSU alignment, 560 in ITS, 883 in LSU,498 in MCM7, 622 in RPB1 and 809 in TEF1. Thecombined dataset included 3876 characters, amongwhich 1123 were parsimony informative, 357 werevariable and parsimony uninformative and 2396 wereconstant. The alignment was deposited in TreeBASE(S14753). A single tree was generated in the MPanalysis. The general time reversible + proportion ofinvariable sites + gamma distributed for rate variationamong sites (GTR+I+G) was selected as the best-fitmodel for BI and ML analyses. The topologies of BIand MP trees were similar to the ML tree and only theML tree is illustrated (FIG. 1).

There two major clades were in the phylogeny(FIG. 1), clade A with 14 species and clade B withthree species. In clade A three Magnaporthiopsisspecies and one of the new species were grouped assubclade C. Three varieties of the type species ofGaeumannomyces, G. graminis, constituted subcladeD. Three strains of Nakataea oryzae formed subcladeE. The other three new species are in subclade F.

Pyricularia oryzae and P. grisea composed subclade G.In clade B three Ophioceras Sacc. species weregrouped together. Based on the molecular phylogenytogether with morphological, biological and ecolog-ical characteristics, a distinct monophyletic genuswith three new species and a new Magnaporthiopsisspecies are proposed.

TAXONOMY

Pseudophialophora J. Luo & N. Zhang, gen. nov.MycoBank MB807080

Etymology: The generic name refers to the morphologicalsimilarity to Phialophora.

Conidiophores single or branched. Conidiogenouscells phialidic, curved, yellowish to hyaline. Conidiaaggregated in slimy heads, oblong ellipsoidal, straightor slightly curved, aseptate, hyaline, smooth.

Type species: Pseudophialophora eragrostis.Habit: On roots of Poaceae plants.Known distribution: New Jersey, USA.Notes: Our six collections shared many characteris-

tics and formed a monophyletic clade in thephylogenetic tree. They were morphologically similarto Magnaporthiopsis and Gaeumannomyces in Phia-lophora-like conidial states and compressed hyphae incolonies on PDA. Magnaporthiopsis differed fromthem with faster growth rates, straight conidiogenouscells and wider and ovoid shaped conidia (Luo andZhang 2013). Gaeumannomyces differed from them byhaving faster growing colonies, straight conidioge-nous cells and presence of sickle-shaped conidia. Thisgenus is also distinguishable from the true Phialoph-ora, which usually has short and pigmented phialidicconidiogenous cells with a conspicuously flaringcollarette, inhabiting rotten wood and potentiallypathogenic to humans (Cole and Kendrick 1973, deHoog et al. 1999, Gams 2000). Pseudophialophora isthus established.

Pseudophialophora eragrostis J. Luo & N. Zhang, sp.nov. FIG. 2A–D

MycoBank MB807081Etymology: The specific epithet refers to the host’s generic

name.

Colonies on PDA 2.7 cm diam after 7 d in the darkat 25 C, grass green, surface velvety to floccus, aerialmycelium yellowish, reverse pigmented, Cossackgreen. Colonies on CMA 1.6 cm after 7 d in the darkat 25 C, Schedes’s green, aerial mycelium sparse,reverse pigmented, peacock green. Conidiophoressingle or branched. Conidiogenous cells phialidic,curved, yellowish, 3–19 3 2–3.7 mm, 1.5–2.5 mm wideat the base, 0.5–1.2 mm wide near the apex (n 5 50).Conidia aggregated in slimy heads, oblong ellipsoidal,

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LUO ET AL.: NEW MAGNAPORTHACEAE SPECIES 583

straight or slightly curved, aseptate, hyaline, smooth,7.5–10.5 3 2–3.5 mm (n 5 50).

Specimens examined: UNITED STATES. NEW JERSEY:Colliers Mills, N40 04.093, W74 26.598, 42 m. Roots ofEragrostis sp., 30 Aug 2012, J. Luo & N. Zhang CM12m9(HOLOTYPE, RUTPP-CM12m9). UNITED STATES. NEW

JERSEY: Colliers Mills, N40 04.093, W74 26.598, 42 m. Rootsof Poaceae plant, 30 Aug 2012, J. Luo & N. ZhangCM20m5-2.

Notes: Two collections were clustered together anddistinct from the other two species in genusPseudophialophora. It is characterized by slow growing

FIG. 1. The maximum likelihood tree inferred from the combined SSU, ITS, LSU, MCM7, RPB1 and TEF1 sequencedatasets. Branch values ($ 50%) of MP bootstrap proportions (MPBP) are noted above internodes. BI posterior probabilities(BIPP) $ 0.95 are shown as thickened branches.

584 MYCOLOGIA

colonies on PDA and CMA, curved conidiophorescells, oblong ellipsoidal conidia and an endophyticnutrition strategy with Eragrostis grass host andassigned as the type species of the genus.

Pseudophialophora panicorum J. Luo & N. Zhang, sp.nov. FIG. 2E–H

MycoBank MB807082Etymology: The specific epithet refers to the host generic

name.

Colonies on PDA 2.6 cm diam after 7 d in the darkat 25 C, Javel green, surface velvety, aerial myceliumyellowish, reverse pigmented, oil green. Colonies onCMA 3.0 cm after 7 d in the dark at 25 C, pale green-yellow, aerial mycelium sparse, reverse pigmented,pale greenish yellow. Conidiophores single orbranched. Conidiogenous cells phialidic, hyaline,curved, 4–22.5 3 1.5–2.7 mm, 1.2–2.2 mm wide at thebase, 0.5–1.2 mm wide near the apex (n 5 50). Conidiaaggregated in slimy heads, oblong ellipsoidal, aseptate,hyaline, smooth, 7.5–10.5 3 2.5–3.5 mm (n 5 50).

Specimens examined: UNITED STATES. NEW JERSEY:Colliers Mills, N40 04.093, W74 26.598, 42 m. Roots of

Poaceae plant, 30 Aug 2012, J. Luo & N. Zhang CM3m7(HOLOTYPE, RUTPP-CM3m7). UNITED STATES. NEWJERSEY: Colliers Mills, N40 04.093, W74 26.598, 42 m. Rootsof Panicum sp., 30 Aug 2012, J. Luo & N. Zhang CM9s6.

Notes: Two collections of this species occurred atthe base of subclade F. Compared to Pseudophilophoraeragrostis, they differed by having lighter colonies onPDA and Panicum host.

Pseudophialophora schizachyrii J. Luo & N. Zhang,sp. nov. FIG. 2I–L

MycoBank MB807083Etymology: The specific epithet refers to the host generic

name.

Colonies on PDA 2.2 cm diam after 7 d in the darkat 25 C, sulphine yellow, surface velvety, aerialmycelium yellowish, reverse pigmented, orange cit-rine. Colonies on CMA reaching 1.8 cm after 7 d inthe dark at 25 C, aniline yellow, aerial myceliumsparse, reverse pigmented, pyrite yellow. Conidio-phores single or branched. Conidiogenous cellsphialidic, hyaline to yellowish, curved, 3–23 3 2.5–3.7 mm, 1.2–2 mm wide at the base, 0.5–1.2 mm widenear the apex (n 5 50). Conidia aggregated in slimyheads, oblong ellipsoidal to ellipsoidal, aseptate,hyaline, smooth, 5.5–8 3 2.5–3.5 mm (n 5 50).

Specimens examined: UNITED STATES. NEW JERSEY:Assunpink Lake, N40 12.962, W74 30.527, 40 m. Roots ofPoaceae plant, 30 Aug 2012, J. Luo & N. Zhang AL3s4(HOLOTYPE, RUTPP-AL3s4). UNITED STATES. NEWJERSEY: Assunpink Lake, N40 12.962, W74 30.527, 40 m.Roots of Schizachyrium sp., 30 Aug 2012, J. Luo & N. ZhangAL2m1.

Notes: This species appeared to be more closelyrelated to Pseudophilophora eragrostis than P. pani-corum in the phylogenetic tree. Common charactersof the genus could be found in all these species,however P. eragrostis differed from P. schizachyii byhaving green colonies, longer conidia and Eragrostishost. P. panicorum differed in greenish colonies,longer conidia and Panicum host.

Magnaporthiopsis panicorum J. Luo & N. Zhang, sp.nov. FIG. 2M–P

MycoBank MB807084Etymology: The specific epithet refers to the host generic

name.

Colonies on PDA 4.8 cm diam after 7 d in the darkat 25 C, parrot green, surface floccus, aerial myceliumyellowish, reverse pigmented, cedar green. Colonieson CMA reaching 5.5 cm after 7 d in the dark at 25 C,pale yellow-green, aerial mycelium sparse, reversepigmented, pale yellow-green. Conidiophores singleor branched. Conidiogenous cells phialidic, hyaline,straight or slightly curved, 5–30.5 3 2–3.5 mm 1.7–

FIG. 2. Conidiophores and conidia. A–D. Pseudophialo-phora eragrostis. E–H. Pseudophialophora panicorum. I–L.Pseudophialophora schizachyrii. M–P. Magnaporthiopsis pa-nicorum.

LUO ET AL.: NEW MAGNAPORTHACEAE SPECIES 585

2.5 mm wide at the base, 0.5–1.5 mm wide near theapex (n 5 50). Conidia aggregated in slimy heads,ovoid, aseptate, hyaline, smooth, 7.5–11.5 3 3.5–5 mm(n 5 50).

Specimens examined: UNITED STATES. NEW JERSEY:Colliers Mills, N40 04.093, W74 26.598, 42 m. Roots ofPanicum sp., 30 Aug 2012, J. Luo & N. Zhang CM2s8(HOLOTYPE, RUTPP-CM2s8). UNITED STATES. NEWJERSEY: Colliers Mills, N40 04.093, W74 26.598, 42 m.Roots of Panicum sp., 30 Aug 2012, J. Luo & N. ZhangCM7m9. UNITED STATES. NEW JERSEY: Colliers Mills,N40 04.093, W74 26.598, 42 m. Roots of Panicum sp., 30Aug 2012, J. Luo & N. Zhang CM9m11. UNITED STATES.NEW JERSEY: Colliers Mills, N40 04.093, W74 26.598, 42 m.Roots of Panicum sp., 30 Aug 2012, J. Luo & N. ZhangCM10s2.

Notes: Four collections of this species were groupedtogether and situated at the base of subclade C. Fastgrowing colonies with wavy and curling hyphae at themargin, phialidic conidiogenous cells, ovoid conidiaand grass host made this species a good fit inMagnaporthiopsis (Luo and Zhang 2013). Comparedto other species in the genus, M. poae differed from itby having olivaceous brown and faster growingcolonies on PDA (1.3 cm/d at 28–30 C), and smallerconidia (3–8 3 1–3 mm) (Landschoot and Jackson1989b). M. rhizophila differed by having gray-brownto olivacous black and faster growing colonies on PDA(0.8 cm/d at 28 C), wider conidiogenous cells andslightly longer conidia (6–20 3 2–6 mm) (Scott andDeacon 1983). M. incrustans differed by havingolivaceous black and faster growing colonies onPDA (1.4 cm/d at 28–30 C), wider conidiogenouscells (9–15 mm) and smaller conidia (3–6 3 2–3 mm)(Landschoot and Jackson 1989a).

DISCUSSION

Studies have shown that there are three majorlineages in Magnaporthaceae, a family in Sordario-mycetes that includes more than 100 species (Cannon1994, Luo and Zhang 2013). The early diverginglineage includes saprotrophic taxa that usually inhab-it submerged woody substrates, such as Ophiocerasand Pseudohalonectria Minoura & T. Muroi. The riceblast pathogen Pyricularia oryzae (Magnaporthe ory-zae) and the gray leaf spot fungus Pyricularia grisea(Magnaporthe grisea) constitute the second lineage,which produce leaf-infecting sympodial conidia. Moremultilocus sequence data is needed to test whetherother Pyricularia Sacc. species also belong to thislineage. The third lineage mainly is composed ofgrass root associated fungi, some of which are root-infecting pathogens, such as Gaeumannomyces grami-nis (take-all pathogen of cereals) and Magnaporthiop-sis poae (summer patch pathogen of turfgrass). The

asexual states of fungi in the third lineage arePhialophora-like or Harpophora-like, with the excep-tion of Nakataea oryzae (Magnaporthe salvinii), whichproduces aerial infecting, sympodial conidia. Thefour new species proposed here belong in the thirdlineage, which is supported by their grass root-association habit, Phialophora-like conidial morphol-ogy and six-locus phylogenetic analysis.

Eighty-three are names in Phialophora (http://www.speciesfungorum.org/), which are poorly differ-entiated by morphology but highly divergent basedon molecular phylogenetic analyses (Gams 2000,Harrington and McNew 2003, Vijaykrishna et al.2004). The true Phialophora, including the typespecies P. verrucosa and its relatives, is linked toCapronia Sacc. in Herpotrichiellaceae of Chaetothyr-iales (Yan et al. 1995, Untereiner and Naveau 1999, deHoog 1999). Based on phylogenetic studies (Gams2000, Harrington and McNew 2003, Vijaykrishna et al.2004, Thongkantha et al. 2009), some Phialophoraspecies have been transferred or placed in othergenera, such as Cadophora Lagerb. & Melin (Derma-teaceae, Helotiales), Harpophora (Magnaporthaceae,Magnaporthales), Lecythophora Nannf. (Coniochaeta-ceae, Coniochaetales), Phaeoacremonium W. Gams,Crous & M.J. Wingf. (Togniniaceae, Diaporthales)and Pleurostomophora Vijaykr., L. Mostert, Jeewon,W. Gams, K.D. Hyde & Crous (Pleurostomataceae,Calosphaeriales).

In Magnaporthaceae, Buergenerula, Ceratosphaeria,Gaeumannomyces and Magnaporthiopsis were reportedto have Phialophora-like or Harpophora-like asexualstates (Cannon 1994, Reblova 2006, Huhndorf et al.2008, Zhang et al. 2011, Luo and Zhang 2013). Oursix-gene phylogenetic analysis indicates that Magna-porthiopsis (with Phialophora-like anamorphs) is asister genus of Gaeumannomyces graminis (withHarpophora anamorphs). Buergenerula spartinae, theonly species with a Harpophora-like anamorph in thegenus, appeared to be closely related to Magna-porthiopsis and Gaeumannomyces graminis. Gaeuman-nomyces cylindrosporus (with Harpophora anamorph)formed a distinct lineage. Ceratosphaeria lampado-phora, the type species of the genus, and C. phialidicaalso produces Harpophora-like anamorphs (Reblova2006, Huhndorf et al. 2008). Previous LSU and SSUrDNA sequence analyses suggested that it was close toPseudohalonectria (Huhndorf et al. 2008, Thong-kantha et al. 2009). Taken together, species inMagnaporthaceae that produce Harpophora-like co-nidia are polyphyletic. The curved conidium mor-phology apparently evolved multiple times in thisfamily. The three species in the proposed new genusPseudophialophora formed a well supported mono-phyletic clade, with common features such as curved

586 MYCOLOGIA

conidiogenous cells without a conspicuous collaretteat the apex that distinguish them from otherPhialophora-like taxa.

Many species in Magnaporthaceae are importantpathogens of cereals and grasses (Scott and Deacon1983; Landschoot and Jackson 1989a, b; Besi et al.2009), but non-pathogenic members do not causedisease symptoms on their hosts. Studies have shownthat nonpathogenic or weakly pathogenic Magna-porthaceae fungi can be used to suppress cerealdiseases caused by other pathogens (Deacon 1974,Speakman 1984, Ulrich et al. 2000, Gutteridge et al.2007). For example, Gaeumannomyces cylindrosporus(Harpophora graminicola) and G. graminis var.graminis may be used as antagonists of G. graminisvar. tritici to control the take-all disease of wheat(Slope et al. 1978, Speakman 1984, Gutteridge et al.2007). The new species reported here apparently didnot cause disease symptoms on the grass hosts andmight be of value in biological control of plantdiseases. Greenhouse inoculations on rice, switch-grass and other plants are needed to test thepathogenicity of these fungi to further evaluate theirnutritional mode and life cycle.

ACKNOWLEDGMENT

The research was financially supported by the NationalScience Foundation (grant DEB 1145174) to Zhang.

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