Mycologia, 106(3), 2014, pp. 580–588. DOI: 10.3852/13-306 # 2014 by The Mycological Society of America, Lawrence, KS 66044-8897
Four new species in Magnaporthaceae from grass roots in New Jersey Pine Barrens Jing Luo Emily Walsh Ning Zhang1
nected to, Buergenerula Syd., Ceratosphaeria Niessl and Gaeumannomyces Arx & D.L. Olivier, while Phialophora-like state was found in Magnaporthiopsis J. Luo & N. Zhang (Cannon 1994, Re´blova´ 2006, Huhndorf et al. 2008, Zhang et al. 2011, Luo and Zhang 2013). In this study 10 Phialophora-like fungal isolates were found during our recent survey of fungi associated with grass roots in New Jersey Pine Barrens. Based on morphology, biology, ecology and multigene phylogenetic analyses, a new genus and four new species are proposed in family Magnaporthaceae.
Department of Plant Biology and Pathology, 201 Foran Hall, 59 Dudley Road, Rutgers University, New Brunswick, New Jersey 08901
Abstract: Based on morphology and DNA sequences of SSU, ITS, LSU, MCM7, RPB1 and TEF1 genes, we describe four new species in Magnaporthaceae that are associated with grass roots collected from New Jersey Pine Barrens. A new genus, Pseudophialophora, is erected to accommodate three species, which is characterized by slow growth on potato dextrose agar, curved conidiogenous cells without a conspicuous collarette at the apex and oblong ellipsoidal conidia. Pseudophialophora eragrostis, P. panicorum and P. schizachyrii are assigned to this genus. A new species of Magnaporthiopsis also is reported and named as M. panicorum. Distinctions between them and phylogenetic relationships with other Magnaporthaceae taxa are discussed. Key words: Harpophora, Magnaporthe, multigene phylogeny, Phialophora, Pseudophialophora, Pyricularia, systematics, taxonomy
MATERIALS AND METHODS Fungal isolation.—Healthy grass roots were sampled from Colliers Mills (N40 04.093, W74 26.598) and Assunpink Lake (N40 12.962, W74 30.527) in the New Jersey Pine Barrens in Aug 2012. Grass samples were transported to the laboratory and processed for fungal isolation within 24 h. The roots were rinsed in tap water to remove soil particles on the surface and cut into ca. 5 mm long fragments. These fragments were surface-sterilized with 75% alcohol for 5 min, followed by 5 min in 0.6% sodium hypochlorite and two final rinses in sterile distilled water (Zhang et al. 2011). The disinfected fragments were placed on malt extract agar (MEA, BD) with 0.07% lactic acid and incubated at room temperature. Fungal cultures were isolated and purified by subculturing from emergent hyphal tips.
INTRODUCTION Phialophora Medlar is a hyphomycetous genus and typified by Phialophora verrucosa Medlar (Medlar 1915). The main characteristics of the genus are darkly pigmented hyphae, simple or branched conidiophores and phialidic conidiogenous cells with a flaring collarette at the apex (Cain 1952, Barnett and Hunter 2006). It is highly polyphyletic and found to be connected to a number of teleomorphic taxa in various orders, such as Caliciales, Chaetothyriales, Diaporthales, Dothideales, Hypocreales, Leotiales, Magnaporthales, Ophiostomatales, Sordariales and Spathulosporales (Gams 2000). Gams (2000) introduced a new asexual genus, Harpophora W. Gams, to accommodate four Phialophora-like species characterized by fast growing colonies, pigmented phialides with a typical collarette, and strongly curved conidia that were reluctant to germinate on standard media. In Magnaporthaceae, Harpophora-like state was con-
Cultural study.— Cultural characteristics were recorded from potato dextrose agar (PDA, BD) and cornmeal agar (CMA, BD), and the color names of colonies followed Ridgway’s nomenclature (Ridgway 1912). Microscopic examinations, measurements and images were taken from slides of fungi mounted in distilled water. The specimens examined were deposited in the Rutgers Mycological Herbarium, New Brunswick, New Jersey (RUTPP), and cultures were deposited in the Centraalbureau voor Schimmelcultures Fungal Biodiversity Centre, the Netherlands (CBS). DNA extraction, amplification and sequence analysis.—The protocols described by Zhang et al. (2011) and Luo and Zhang (2013) were used for DNA extraction, PCR amplification and sequencing of small subunit (SSU), internal transcribed spacer (ITS), and large subunit (LSU) of ribosomal RNA genes, DNA replication licensing factor (MCM7), the largest subunit of RNA polymerase II (RPB1) and translation elongation factor 1-a (TEF1) genes. In this study 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: [email protected]
580
LUO ET AL.: NEW MAGNAPORTHACEAE SPECIES with the isolate numbers, sources, hosts and GenBank accession 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). Sixgene datasets were assembled for phylogenetic analyses. Cryphonectria parasitica was used as outgroup taxon. Maximum likelihood (ML) analysis with the selected model was carried out in PAUP* 4.0b10 (Swofford 2002). Starting trees were obtained by random sequence addition with 100 replicates in heuristic search. The branch swapping algorithm was tree-bisection-reconnection (TBR), and both steepest descent and MULTREES options were not in effect. Maximum parsimony (MP) analysis was performed with heuristic search in PAUP* 4.0b10 (Swofford 2002). All characters were given equal weight. Gaps were treated as missing characters. Starting trees were obtained via random sequence stepwise addition with 1000 replicates. The branch-swapping algorithm was tree-bisection-reconnection (TBR). Steepest descent and MULTREES options were not in effect. To calculate all branch support values, a bootstrap analysis was performed with 1000 replicates using heuristic searches with simple sequence stepwise addition for each replicate. Bayesian inference (BI) was conducted with the Markov chain Monte Carlo method in MrBayes 3.2.1 (Ronquist et al. 2012) under the nucleotide substitution model selected by using hierarchical likelihood ratio tests (hLRTs) and Akaike information criterion (AIC) in MrModeltest 2.3 (Nylander 2004). Trees were sampled every 100 generations from 10 000 000 generations resulting in 100 000 trees. The first 25 000 trees were discarded as burn-in and the remaining 75 000 trees were chosen to calculate posterior probability values of clades in a consensus tree.
RESULTS A total of 504 nucleotide characters including gaps were in the SSU alignment, 560 in ITS, 883 in LSU, 498 in MCM7, 622 in RPB1 and 809 in TEF1. The combined dataset included 3876 characters, among which 1123 were parsimony informative, 357 were variable and parsimony uninformative and 2396 were constant. The alignment was deposited in TreeBASE (S14753). A single tree was generated in the MP analysis. The general time reversible + proportion of invariable sites + gamma distributed for rate variation among sites (GTR+I+G) was selected as the best-fit model for BI and ML analyses. The topologies of BI and MP trees were similar to the ML tree and only the ML tree is illustrated (FIG. 1). There two major clades were in the phylogeny (FIG. 1), clade A with 14 species and clade B with three species. In clade A three Magnaporthiopsis species and one of the new species were grouped as subclade C. Three varieties of the type species of Gaeumannomyces, G. graminis, constituted subclade D. Three strains of Nakataea oryzae formed subclade E. The other three new species are in subclade F.
581
Pyricularia oryzae and P. grisea composed subclade G. In clade B three Ophioceras Sacc. species were grouped together. Based on the molecular phylogeny together with morphological, biological and ecological characteristics, a distinct monophyletic genus with three new species and a new Magnaporthiopsis species are proposed. TAXONOMY Pseudophialophora J. Luo & N. Zhang, gen. nov. MycoBank MB807080 Etymology: The generic name refers to the morphological similarity to Phialophora.
Conidiophores single or branched. Conidiogenous cells phialidic, curved, yellowish to hyaline. Conidia aggregated in slimy heads, oblong ellipsoidal, straight or 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 characteristics and formed a monophyletic clade in the phylogenetic tree. They were morphologically similar to Magnaporthiopsis and Gaeumannomyces in Phialophora-like conidial states and compressed hyphae in colonies on PDA. Magnaporthiopsis differed from them with faster growth rates, straight conidiogenous cells and wider and ovoid shaped conidia (Luo and Zhang 2013). Gaeumannomyces differed from them by having faster growing colonies, straight conidiogenous cells and presence of sickle-shaped conidia. This genus is also distinguishable from the true Phialophora, which usually has short and pigmented phialidic conidiogenous cells with a conspicuously flaring collarette, inhabiting rotten wood and potentially pathogenic to humans (Cole and Kendrick 1973, de Hoog et al. 1999, Gams 2000). Pseudophialophora is thus established. Pseudophialophora eragrostis J. Luo & N. Zhang, sp. nov. FIG. 2A–D MycoBank MB807081 Etymology: The specific epithet refers to the host’s generic name.
Colonies on PDA 2.7 cm diam after 7 d in the dark at 25 C, grass green, surface velvety to floccus, aerial mycelium yellowish, reverse pigmented, Cossack green. Colonies on CMA 1.6 cm after 7 d in the dark at 25 C, Schedes’s green, aerial mycelium sparse, reverse pigmented, peacock green. Conidiophores single or branched. Conidiogenous cells phialidic, curved, yellowish, 3–19 3 2–3.7 mm, 1.5–2.5 mm wide at the base, 0.5–1.2 mm wide near the apex (n 5 50). Conidia aggregated in slimy heads, oblong ellipsoidal,
M71
Lisa9 PRR1-4756
FF9
GI111 ATCC 44754
M47 M22
M23 M35
M51 M21
Ggt568
M55 CM2s8
73-15
GggFL64 GggFL199
M53 M54 CBS 235.32 R3-111a-1
CM7m9 CM9m11 CM10s2 ATCC 64411
Florida, USA
GgFL199
M33
Glenn County, California, USA Glenn County, California, USA
Kansas, USA Japan
unknown unknown
New Jersey, USA unknown
New Jersey, USA New Jersey, USA New Jersey, USA USA
Montana, USA New Jersey, USA
Florida, USA Florida, USA Arkansas, USA Washington, USA
the Netherlands
ATCC 38755
Host MCM7
RPB1
TEF1
JF710374
JF414896
JF710392
JF710442
JF710411
JF414895 JF414887
JF414883 JF414892
JF414885 JF414882
JF710389 JF710382
JF710384 JF710386
JF710390 JF710383
JF710440 JF710441
JF710432 JF710437
JF710433 JF710431
JF710417 JF710406
JF710408 JF710412
JF710415 JF710407
JX134659 JX134673 JX134685 JX134713 JX134727 JX134699
JX134658 JX134672 JX134684 JX134712 JX134726 JX134698
JF414846 JF414838
JF414834 JF414843
JF414836 JF414833
KF689595 KF689645 KF689635 KF689605 KF689615 KF689625 KF689596 KF689646 KF689636 KF689606 KF689616 KF689626 KF689594 KF689644 KF689634 KF689604 KF689614 KF689624 Genome data, Broad Institute
JF414875 JF414850 JF414900 JF710395 JF710445 JF710420 KF689593 KF689643b KF689633 KF689603 KF689613 KF689623
JF414872 JF414847 JF414897 JF710393 JF710443 JF710418 JF414873 JF414848 JF414898 JF710394 JF710444 JF710419 DQ341476 JX134669 JX134681 JX134709 JX134723 JX134695 Broad Institute
JF414871
JX134655 JX134668 JX134680 JX134708 JX134722 JX134694
DQ341473 JX134667 DQ341494 JX134707 JX134721 JX134693
Zoysia matrella JF414870 Oryza sativa JF414862
Oryza sp.
LSU
Genome data, Joint Genome Institute
Poa pratensis JF414858 unknown JF414867
Oryza sp.
ITS
DQ341471 JX134666 DQ341492 JX134706 JX134720 JX134692
SSU
Poa pratensis JF414860 unknown JF414857
Panicum sp. Panicum sp. Panicum sp. Triticum sp.
Triticum sp. Panicum sp.
Stenotaphrum secundatum unknown unknown Oryza sativa Genome data,
Avena sativa
Spartina alterniflora Connecticut, USA Castanea dentata UK Grass root
unknown
Source
CBS 187.65
CBS 610.75
EP155
N. oryzae
ATCC 22848
Isolate no.a Alternative no.
M69
Species
Species name, isolate number, alternative number, source, host and GenBank accession numbers of the fungi used in this study
Buergenerula spartinae Kohlm. & R.V. Gessner Cryphonectria parasitica (Murrill) M.E. Barr Gaeumannomyces cylindrosporus D. Hornby, Slope, Gutter. & Sivan. G. graminis var. avenae (E.M. Turner) Dennis G. graminis var. graminis (Sacc.) Arx & D.L. Olivier G. graminis var. graminis G. graminis var. graminis G. graminis var. graminis G. graminis var. tritici J. Walker G. graminis var. tritici Magnaporthiopsis panicorum J. Luo & N. Zhang M. panicorum M. panicorum M. panicorum M. poae (Landsch. & N. Jacks.) J. Luo & N. Zhang M. poae M. rhizophila (D.B. Scott & Deacon) J. Luo & N. Zhang M. rhizophila M. incrustans (Landsch. & N. Jacks.) J. Luo & N. Zhang M. incrustans Nakataea oryzae (Catt.) J. Luo & N. Zhang N. oryzae
TABLE I.
582 MYCOLOGIA
New Jersey, USA New Jersey, USA
New Jersey, USA CG-14 CG-4
CM9s6 AL3s4
AL2m1 M82 M83 70-15 M25 M60
Pyricularia grisea Sacc.
P. grisea
P. oryzae Cavara
P. oryzae P. oryzae
unknown New Jersey, USA
ITS
LSU MCM7
RPB1
TEF1
KF689601 KF689651 KF689641 KF689611 KF689621 KF689631 KF689600 KF689650 KF689640 KF689610 KF689620 KF689630
KF689597 KF689647 KF689637 KF689607 KF689617 KF689627 KF689602 KF689652 KF689642 KF689612 KF689622 KF689632
JF414839 JF414840
JF414888 JF414889
Genome data, Broad Institute JF710397 JF710398
JF710449 JF710447
JF710422 JF710423
JX134657 JX134671 JX134683 JX134711 JX134725 JX134697
Oryza sativa JF414863 Festuca JF414864 arundinacea
Oryza sativa
Digitaria sp.
Schizachyrium KF689599 KF689649 KF689639 KF689609 KF689619 KF689629 sp. Digitaria sp. JX134656 JX134670 JX134682 JX134710 JX134724 JX134696
Panicum sp. Poaceae sp.
Poaceae sp. Poaceae sp.
Eragrostis sp. KF689598 KF689648 KF689638 KF689608 KF689618 KF689628
JX134665 JX134679 JX134691 JX134719 JX134733 JX134705
JX134662 JX134676 JX134688 JX134716 JX134730 JX134702 JX134663 JX134677 JX134689 JX134717 JX134731 JX134703 JX134664 JX134678 JX134690 JX134718 JX134732 JX134704
a ATCC 5 American Type Culture Collection, Manassas, Virginia; CBS 5 Centraalbureau voor Schimmelcultures, Utrecht, the Netherlands; HKUCC 5 The University of Hong Kong Culture Collection, Hong Kong; YMF 5 the Yunnan Microbiological Fermentation Culture Collection Center, Yunnan, China. b Numbers in boldface indicate newly submitted sequences.
ATCC MYA4617 IG1, Pg rough 223S
New Jersey, USA New Jersey, USA
CM20m5-2 CM3m7
Tichnor, Arkansas, USA Tichnor, Arkansas, USA unknown
New Jersey, USA
YMF1.01288
M95
SSU
Panicum JX134660 JX134674 JX134686 JX134714 JX134728 JX134700 effusum var. effusum rotten wood JX134661 JX134675 JX134687 JX134715 JX134729 JX134701
Host
Yunnan, China rotten wood Hong Kong, China rotten wood Exeter, UK dead stem of dicot plant, probably Urtica dioica Yunnan, China rotten wood
CM12m9
YMF1.00981 HKUCC 3936 ATCC 24161
M92 CBS 114926 CBS 894.70
Yunnan, China
Queensland, Australia
Source
Pseudohalonectria lignicola Minoura & T. Muroi Pseudophialophora eragrostis J. Luo & N. Zhang P. eragrostis Pseudophialophora panicorum J. Luo & N. Zhang P. panicorum Pseudophialophora schizachyrii J. Luo & N. Zhang P. schizachyrii
YMF1.00980
M91
Ophioceras commune Shearer, J.L. Crane & W. Chen O. commune O. dolichostomum O. leptosporum (S.H. Iqbal) J. Walker
Isolate no.a Alternative no. ATCC 200212
Species
Continued
Omnidemptus affinis P.F. Cannon & Alcorn
TABLE I.
LUO ET AL.: NEW MAGNAPORTHACEAE SPECIES 583
584
MYCOLOGIA
FIG. 1. The maximum likelihood tree inferred from the combined SSU, ITS, LSU, MCM7, RPB1 and TEF1 sequence datasets. Branch values ($ 50%) of MP bootstrap proportions (MPBP) are noted above internodes. BI posterior probabilities (BIPP) $ 0.95 are shown as thickened branches.
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 of Eragrostis 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. Roots of Poaceae plant, 30 Aug 2012, J. Luo & N. Zhang CM20m5-2.
Notes: Two collections were clustered together and distinct from the other two species in genus Pseudophialophora. It is characterized by slow growing
LUO ET AL.: NEW MAGNAPORTHACEAE SPECIES
585
Poaceae plant, 30 Aug 2012, J. Luo & N. Zhang CM3m7 (HOLOTYPE, RUTPP-CM3m7). UNITED STATES. NEW JERSEY: Colliers Mills, N40 04.093, W74 26.598, 42 m. Roots of Panicum sp., 30 Aug 2012, J. Luo & N. Zhang CM9s6.
Notes: Two collections of this species occurred at the base of subclade F. Compared to Pseudophilophora eragrostis, they differed by having lighter colonies on PDA and Panicum host. Pseudophialophora schizachyrii J. Luo & N. Zhang, sp. nov. FIG. 2I–L MycoBank MB807083 Etymology: The specific epithet refers to the host generic name.
Colonies on PDA 2.2 cm diam after 7 d in the dark at 25 C, sulphine yellow, surface velvety, aerial mycelium yellowish, reverse pigmented, orange citrine. Colonies on CMA reaching 1.8 cm after 7 d in the dark at 25 C, aniline yellow, aerial mycelium sparse, reverse pigmented, pyrite yellow. Conidiophores single or branched. Conidiogenous cells phialidic, 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 wide near the apex (n 5 50). Conidia aggregated in slimy heads, oblong ellipsoidal to ellipsoidal, aseptate, hyaline, smooth, 5.5–8 3 2.5–3.5 mm (n 5 50). FIG. 2. Conidiophores and conidia. A–D. Pseudophialophora eragrostis. E–H. Pseudophialophora panicorum. I–L. Pseudophialophora schizachyrii. M–P. Magnaporthiopsis panicorum.
colonies on PDA and CMA, curved conidiophores cells, oblong ellipsoidal conidia and an endophytic nutrition strategy with Eragrostis grass host and assigned as the type species of the genus. Pseudophialophora panicorum J. Luo & N. Zhang, sp. nov. FIG. 2E–H MycoBank MB807082 Etymology: The specific epithet refers to the host generic name.
Colonies on PDA 2.6 cm diam after 7 d in the dark at 25 C, Javel green, surface velvety, aerial mycelium yellowish, reverse pigmented, oil green. Colonies on CMA 3.0 cm after 7 d in the dark at 25 C, pale greenyellow, aerial mycelium sparse, reverse pigmented, pale greenish yellow. Conidiophores single or branched. Conidiogenous cells phialidic, hyaline, curved, 4–22.5 3 1.5–2.7 mm, 1.2–2.2 mm wide at the base, 0.5–1.2 mm wide near the apex (n 5 50). Conidia aggregated 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
Specimens examined: UNITED STATES. NEW JERSEY: Assunpink Lake, N40 12.962, W74 30.527, 40 m. Roots of Poaceae plant, 30 Aug 2012, J. Luo & N. Zhang AL3s4 (HOLOTYPE, RUTPP-AL3s4). UNITED STATES. NEW JERSEY: Assunpink Lake, N40 12.962, W74 30.527, 40 m. Roots of Schizachyrium sp., 30 Aug 2012, J. Luo & N. Zhang AL2m1.
Notes: This species appeared to be more closely related to Pseudophilophora eragrostis than P. panicorum in the phylogenetic tree. Common characters of the genus could be found in all these species, however P. eragrostis differed from P. schizachyii by having green colonies, longer conidia and Eragrostis host. P. panicorum differed in greenish colonies, longer conidia and Panicum host. Magnaporthiopsis panicorum J. Luo & N. Zhang, sp. nov. FIG. 2M–P MycoBank MB807084 Etymology: The specific epithet refers to the host generic name.
Colonies on PDA 4.8 cm diam after 7 d in the dark at 25 C, parrot green, surface floccus, aerial mycelium yellowish, reverse pigmented, cedar green. Colonies on CMA reaching 5.5 cm after 7 d in the dark at 25 C, pale yellow-green, aerial mycelium sparse, reverse pigmented, pale yellow-green. Conidiophores single or branched. Conidiogenous cells phialidic, hyaline, straight or slightly curved, 5–30.5 3 2–3.5 mm 1.7–
586
MYCOLOGIA
2.5 mm wide at the base, 0.5–1.5 mm wide near the apex (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 of Panicum sp., 30 Aug 2012, J. Luo & N. Zhang CM2s8 (HOLOTYPE, RUTPP-CM2s8). UNITED STATES. NEW JERSEY: Colliers Mills, N40 04.093, W74 26.598, 42 m. Roots of Panicum sp., 30 Aug 2012, J. Luo & N. Zhang CM7m9. UNITED STATES. NEW JERSEY: Colliers Mills, N40 04.093, W74 26.598, 42 m. Roots of Panicum sp., 30 Aug 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. Zhang CM10s2.
Notes: Four collections of this species were grouped together and situated at the base of subclade C. Fast growing colonies with wavy and curling hyphae at the margin, phialidic conidiogenous cells, ovoid conidia and grass host made this species a good fit in Magnaporthiopsis (Luo and Zhang 2013). Compared to other species in the genus, M. poae differed from it by having olivaceous brown and faster growing colonies on PDA (1.3 cm/d at 28–30 C), and smaller conidia (3–8 3 1–3 mm) (Landschoot and Jackson 1989b). M. rhizophila differed by having gray-brown to olivacous black and faster growing colonies on PDA (0.8 cm/d at 28 C), wider conidiogenous cells and slightly longer conidia (6–20 3 2–6 mm) (Scott and Deacon 1983). M. incrustans differed by having olivaceous black and faster growing colonies on PDA (1.4 cm/d at 28–30 C), wider conidiogenous cells (9–15 mm) and smaller conidia (3–6 3 2–3 mm) (Landschoot and Jackson 1989a). DISCUSSION Studies have shown that there are three major lineages in Magnaporthaceae, a family in Sordariomycetes that includes more than 100 species (Cannon 1994, Luo and Zhang 2013). The early diverging lineage includes saprotrophic taxa that usually inhabit submerged woody substrates, such as Ophioceras and Pseudohalonectria Minoura & T. Muroi. The rice blast pathogen Pyricularia oryzae (Magnaporthe oryzae) and the gray leaf spot fungus Pyricularia grisea (Magnaporthe grisea) constitute the second lineage, which produce leaf-infecting sympodial conidia. More multilocus sequence data is needed to test whether other Pyricularia Sacc. species also belong to this lineage. The third lineage mainly is composed of grass root associated fungi, some of which are rootinfecting pathogens, such as Gaeumannomyces graminis (take-all pathogen of cereals) and Magnaporthiopsis poae (summer patch pathogen of turfgrass). The
asexual states of fungi in the third lineage are Phialophora-like or Harpophora-like, with the exception of Nakataea oryzae (Magnaporthe salvinii), which produces aerial infecting, sympodial conidia. The four new species proposed here belong in the third lineage, which is supported by their grass rootassociation habit, Phialophora-like conidial morphology and six-locus phylogenetic analysis. Eighty-three are names in Phialophora (http:// www.speciesfungorum.org/), which are poorly differentiated by morphology but highly divergent based on molecular phylogenetic analyses (Gams 2000, Harrington and McNew 2003, Vijaykrishna et al. 2004). The true Phialophora, including the type species P. verrucosa and its relatives, is linked to Capronia Sacc. in Herpotrichiellaceae of Chaetothyriales (Yan et al. 1995, Untereiner and Naveau 1999, de Hoog 1999). Based on phylogenetic studies (Gams 2000, Harrington and McNew 2003, Vijaykrishna et al. 2004, Thongkantha et al. 2009), some Phialophora species have been transferred or placed in other genera, such as Cadophora Lagerb. & Melin (Dermateaceae, Helotiales), Harpophora (Magnaporthaceae, Magnaporthales), Lecythophora Nannf. (Coniochaetaceae, 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 reported to have Phialophora-like or Harpophora-like asexual states (Cannon 1994, Re´blova´ 2006, Huhndorf et al. 2008, Zhang et al. 2011, Luo and Zhang 2013). Our six-gene phylogenetic analysis indicates that Magnaporthiopsis (with Phialophora-like anamorphs) is a sister genus of Gaeumannomyces graminis (with Harpophora anamorphs). Buergenerula spartinae, the only species with a Harpophora-like anamorph in the genus, appeared to be closely related to Magnaporthiopsis and Gaeumannomyces graminis. Gaeumannomyces cylindrosporus (with Harpophora anamorph) formed a distinct lineage. Ceratosphaeria lampadophora, the type species of the genus, and C. phialidica also produces Harpophora-like anamorphs (Re´blova´ 2006, Huhndorf et al. 2008). Previous LSU and SSU rDNA sequence analyses suggested that it was close to Pseudohalonectria (Huhndorf et al. 2008, Thongkantha et al. 2009). Taken together, species in Magnaporthaceae that produce Harpophora-like conidia are polyphyletic. The curved conidium morphology apparently evolved multiple times in this family. The three species in the proposed new genus Pseudophialophora formed a well supported monophyletic clade, with common features such as curved
LUO ET AL.: NEW MAGNAPORTHACEAE SPECIES conidiogenous cells without a conspicuous collarette at the apex that distinguish them from other Phialophora-like taxa. Many species in Magnaporthaceae are important pathogens of cereals and grasses (Scott and Deacon 1983; Landschoot and Jackson 1989a, b; Besi et al. 2009), but non-pathogenic members do not cause disease symptoms on their hosts. Studies have shown that nonpathogenic or weakly pathogenic Magnaporthaceae fungi can be used to suppress cereal diseases 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. graminis var. 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 did not cause disease symptoms on the grass hosts and might be of value in biological control of plant diseases. Greenhouse inoculations on rice, switchgrass and other plants are needed to test the pathogenicity of these fungi to further evaluate their nutritional mode and life cycle. ACKNOWLEDGMENT The research was financially supported by the National Science Foundation (grant DEB 1145174) to Zhang.
LITERATURE CITED Barnett HL, Hunter BB. 2006. Illustrated genera of imperfect fungi. 4th ed. St Paul, Minnesota: APS Press, 218 p. Besi MI, Tucker SL, Sesma A. 2009. Magnaporthe and its relatives. In: Encyclopedia of life sciences. Chichester, UK: John Wiley & Sons. p 1–9. doi: 10.1002/ 9780470015902.a0021311 Cain RF. 1952. Studies of fungi imperfecti I. Phialophora. Can J Bot 30:338–343, doi:10.1139/b52-025 Cannon PF. 1994. The newly recognized family Magnaporthaceae and its interrelationships. Syst Ascomycetum 13:25–42. Cole GT, Kendrick WB. 1973. Taxonomic studies of Phialiphora. Mycologia 65:661–688, doi:10.2307/ 3758266 de Hoog GS, Weenink XO, Gerrits van den Ende AHG. 1999. Taxonomy of the Phialophora verrucosa complex with the description of two new species. Stud Mycol 43:107–121. Deacon W. 1974. Interactions between varieties of Gaeumannomyces graminis and Phialophora radicicola on roots, stem bases and rhizomes of the Gramineae. Plant Pathol 23:85–92, doi:10.1111/j.1365-3059.1974.tb02914.x Gams W. 2000. Phialophora and some similar morphologically little-differentiated anamorphs of divergent ascomycetes. Stud Mycol 45:187–199.
587
Gutteridge RJ, Jankyn JF, Bateman GL. 2007. The potential of nonpathogenic Gaeumannomyces spp., occurring naturally or introduced into wheat crops or preceding crops, for controlling take-all in wheat. Ann Appl Biol 150:53–64, doi:10.1111/j.1744-7348.2006.00107.x Hall TA. 1999. Bioedit: a user-friendly biological sequences alignment editor analysis program for windows 95/98/ NT. Nucleic Acids Symp Ser 41:95–98. Harrington TC, McNew DL. 2003. Phylogenetic analysis places the Phialophora-like anamorph genus Cadophora in the Helotiales. Mycotaxon 87:141–151. Huhndorf SM, Greif M, Mugambi GK, Miller AN. 2008. Two new genera in the Magnaporthaceae, a new addition to Ceratosphaeria and two new species of Lentomitella. Mycologia 100:940–955, doi:10.3852/08-037 Landschoot PJ, Jackson N. 1989a. Gaeumannomyces incrustans sp. nov., a root-infecting hyphopodiate fungus from grass roots in the United States. Mycol Res 93:55– 58, doi:10.1016/S0953-7562(89)80136-4 ———, ———. 1989b. Magnaporthe poae sp. nov., a hyphopodiate fungus with a Phialophora anamorph from grass roots in the United States. Mycol Res 93:59– 62, doi:10.1016/S0953-7562(89)80137-6 Luo J, Zhang N. 2013. Magnaporthiopsis, a new genus in Magnaporthaceae. Mycologia 105:1019–1029, doi:10. 3852/12-359 Medlar EM. 1915. A new fungus, Phialophora verrucosa, pathogenic for man. Mycologia 7:200–203, doi:10. 2307/3753363 Nylander JAA. 2004. MrModeltest 2.2. Program distributed by the author. Uppsala, Sweden: Evolutionary Biology Centre. Re´blova´ M. 2006. Molecular systematics of Ceratostomella sensu lato and morphologically similar fungi. Mycologia 98:68–93, doi:10.3852/mycologia.98.1.68 Ridgway R. 1912. Color standards and color nomenclature. Washington DC: Ridgway. Ronquist F, Teslenko M, van der Mark P, Ayres D, Darling A, Ho¨hna S, Larget B, Liu L, Suchard MA, Huelsenbeck JP. 2012. MrBayes 3.2: efficient Bayesian phylogenetic inference and model choice across a large model space. Syst Biol 61:539–542, doi:10.1093/sysbio/sys029 Scott DB, Deacon JW. 1983. Magnaporthe rhizophila sp. nov., a dark mycelial fungus with a Phialophora conidial state, from cereal roots in South Africa. Trans Br Mycol Soc 81:77–81, doi:10.1016/S0007-1536(83)80206-X Slope DB, Salt GA, Broom EW, Gutterodge RJ. 1978. Occurrence of Phialophora radicicola var. graminicola and Gaeumannomyces graminis var. tritici on roots of wheat in field crops. Ann Appl Biol 88:239–246, doi:10.1111/j.1744-7348.1978.tb00701.x Speakman JB. 1984. Control of Gaeumannomyces graminis var. tritici in wheat by isolates of the Gaeumannomyces graminis var. graminis/Phialophora sp. (lobed hyphopodia) complex under field conditions. Phytopathol Z 109:188–191, doi:10.1111/j.1439-0434.1984.tb00706.x Swofford DL. 2002. PAUP* 4b10: phylogenetic analysis using parsimony (*and other methods). Sunderland, Massachusetts: Sinauer Associates. Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgin DG. 1997. The Clustal X Windows interface:
588
MYCOLOGIA
flexible strategies for multiple sequences alignment aided by quality analysis tools. Nucleic Acids Res 25: 4876–4882, doi:10.1093/nar/25.24.4876 Thongkantha S, Jeewon R, Vijaykrishna D, Lumyong S, Mckenzie EHC, Hyde KD. 2009. Molecular phylogeny of Magnaporthaceae (Sordariomycetes) with a new species Ophioceras chiangdaoense from Dracaena loureiroi in Thailand. Fungal Divers 34:157–173. Ulrich K, Augustin C, Werner A. 2000. Identification and characterization of a new group of root-colonizing fungi within the Gaeumannomyces-Phialophora complex. New Phytol 145:127–135, doi:10.1046/j.1469-8137.2000.00553.x Untereiner WA, Naveau FA. 1999. Molecular systematics of the Herpotrichiellaceae with an assessment of the phylogenetic position of Exophiala dermatitidis and
Phialophora americana. Mycologia 91:67–83, doi:10. 2307/3761194 Vijaykrishna D, Mostert L, Jeewon R, GamsW, Hyde KD, Crous PW. 2004. Pleurostomophora, an anamorph of Pleurostoma (Calosphaeriales), a new anamorph genus morphologically similar to Phialophora. Stud Mycol 50: 387–395. Yan ZH, Rogers SO, Wang CJK. 1995. Assessment of Phialophora species based on ribosomal DNA internal transcribed spacers and morphology. Mycologia 87:72– 83, doi:10.2307/3760949 Zhang N, Zhao S, Shen Q. 2011. A six-gene phylogeny reveals the evolution of mode of infection in the rice blast fungus and allied species. Mycologia 103:1267– 1276, doi:10.3852/11-022
Four new species in Magnaporthaceae from grass roots in New Jersey Pine Barrens Jing Luo Emily Walsh Ning Zhang1
nected to, Buergenerula Syd., Ceratosphaeria Niessl and Gaeumannomyces Arx & D.L. Olivier, while Phialophora-like state was found in Magnaporthiopsis J. Luo & N. Zhang (Cannon 1994, Re´blova´ 2006, Huhndorf et al. 2008, Zhang et al. 2011, Luo and Zhang 2013). In this study 10 Phialophora-like fungal isolates were found during our recent survey of fungi associated with grass roots in New Jersey Pine Barrens. Based on morphology, biology, ecology and multigene phylogenetic analyses, a new genus and four new species are proposed in family Magnaporthaceae.
Department of Plant Biology and Pathology, 201 Foran Hall, 59 Dudley Road, Rutgers University, New Brunswick, New Jersey 08901
Abstract: Based on morphology and DNA sequences of SSU, ITS, LSU, MCM7, RPB1 and TEF1 genes, we describe four new species in Magnaporthaceae that are associated with grass roots collected from New Jersey Pine Barrens. A new genus, Pseudophialophora, is erected to accommodate three species, which is characterized by slow growth on potato dextrose agar, curved conidiogenous cells without a conspicuous collarette at the apex and oblong ellipsoidal conidia. Pseudophialophora eragrostis, P. panicorum and P. schizachyrii are assigned to this genus. A new species of Magnaporthiopsis also is reported and named as M. panicorum. Distinctions between them and phylogenetic relationships with other Magnaporthaceae taxa are discussed. Key words: Harpophora, Magnaporthe, multigene phylogeny, Phialophora, Pseudophialophora, Pyricularia, systematics, taxonomy
MATERIALS AND METHODS Fungal isolation.—Healthy grass roots were sampled from Colliers Mills (N40 04.093, W74 26.598) and Assunpink Lake (N40 12.962, W74 30.527) in the New Jersey Pine Barrens in Aug 2012. Grass samples were transported to the laboratory and processed for fungal isolation within 24 h. The roots were rinsed in tap water to remove soil particles on the surface and cut into ca. 5 mm long fragments. These fragments were surface-sterilized with 75% alcohol for 5 min, followed by 5 min in 0.6% sodium hypochlorite and two final rinses in sterile distilled water (Zhang et al. 2011). The disinfected fragments were placed on malt extract agar (MEA, BD) with 0.07% lactic acid and incubated at room temperature. Fungal cultures were isolated and purified by subculturing from emergent hyphal tips.
INTRODUCTION Phialophora Medlar is a hyphomycetous genus and typified by Phialophora verrucosa Medlar (Medlar 1915). The main characteristics of the genus are darkly pigmented hyphae, simple or branched conidiophores and phialidic conidiogenous cells with a flaring collarette at the apex (Cain 1952, Barnett and Hunter 2006). It is highly polyphyletic and found to be connected to a number of teleomorphic taxa in various orders, such as Caliciales, Chaetothyriales, Diaporthales, Dothideales, Hypocreales, Leotiales, Magnaporthales, Ophiostomatales, Sordariales and Spathulosporales (Gams 2000). Gams (2000) introduced a new asexual genus, Harpophora W. Gams, to accommodate four Phialophora-like species characterized by fast growing colonies, pigmented phialides with a typical collarette, and strongly curved conidia that were reluctant to germinate on standard media. In Magnaporthaceae, Harpophora-like state was con-
Cultural study.— Cultural characteristics were recorded from potato dextrose agar (PDA, BD) and cornmeal agar (CMA, BD), and the color names of colonies followed Ridgway’s nomenclature (Ridgway 1912). Microscopic examinations, measurements and images were taken from slides of fungi mounted in distilled water. The specimens examined were deposited in the Rutgers Mycological Herbarium, New Brunswick, New Jersey (RUTPP), and cultures were deposited in the Centraalbureau voor Schimmelcultures Fungal Biodiversity Centre, the Netherlands (CBS). DNA extraction, amplification and sequence analysis.—The protocols described by Zhang et al. (2011) and Luo and Zhang (2013) were used for DNA extraction, PCR amplification and sequencing of small subunit (SSU), internal transcribed spacer (ITS), and large subunit (LSU) of ribosomal RNA genes, DNA replication licensing factor (MCM7), the largest subunit of RNA polymerase II (RPB1) and translation elongation factor 1-a (TEF1) genes. In this study 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: [email protected]
580
LUO ET AL.: NEW MAGNAPORTHACEAE SPECIES with the isolate numbers, sources, hosts and GenBank accession 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). Sixgene datasets were assembled for phylogenetic analyses. Cryphonectria parasitica was used as outgroup taxon. Maximum likelihood (ML) analysis with the selected model was carried out in PAUP* 4.0b10 (Swofford 2002). Starting trees were obtained by random sequence addition with 100 replicates in heuristic search. The branch swapping algorithm was tree-bisection-reconnection (TBR), and both steepest descent and MULTREES options were not in effect. Maximum parsimony (MP) analysis was performed with heuristic search in PAUP* 4.0b10 (Swofford 2002). All characters were given equal weight. Gaps were treated as missing characters. Starting trees were obtained via random sequence stepwise addition with 1000 replicates. The branch-swapping algorithm was tree-bisection-reconnection (TBR). Steepest descent and MULTREES options were not in effect. To calculate all branch support values, a bootstrap analysis was performed with 1000 replicates using heuristic searches with simple sequence stepwise addition for each replicate. Bayesian inference (BI) was conducted with the Markov chain Monte Carlo method in MrBayes 3.2.1 (Ronquist et al. 2012) under the nucleotide substitution model selected by using hierarchical likelihood ratio tests (hLRTs) and Akaike information criterion (AIC) in MrModeltest 2.3 (Nylander 2004). Trees were sampled every 100 generations from 10 000 000 generations resulting in 100 000 trees. The first 25 000 trees were discarded as burn-in and the remaining 75 000 trees were chosen to calculate posterior probability values of clades in a consensus tree.
RESULTS A total of 504 nucleotide characters including gaps were in the SSU alignment, 560 in ITS, 883 in LSU, 498 in MCM7, 622 in RPB1 and 809 in TEF1. The combined dataset included 3876 characters, among which 1123 were parsimony informative, 357 were variable and parsimony uninformative and 2396 were constant. The alignment was deposited in TreeBASE (S14753). A single tree was generated in the MP analysis. The general time reversible + proportion of invariable sites + gamma distributed for rate variation among sites (GTR+I+G) was selected as the best-fit model for BI and ML analyses. The topologies of BI and MP trees were similar to the ML tree and only the ML tree is illustrated (FIG. 1). There two major clades were in the phylogeny (FIG. 1), clade A with 14 species and clade B with three species. In clade A three Magnaporthiopsis species and one of the new species were grouped as subclade C. Three varieties of the type species of Gaeumannomyces, G. graminis, constituted subclade D. Three strains of Nakataea oryzae formed subclade E. The other three new species are in subclade F.
581
Pyricularia oryzae and P. grisea composed subclade G. In clade B three Ophioceras Sacc. species were grouped together. Based on the molecular phylogeny together with morphological, biological and ecological characteristics, a distinct monophyletic genus with three new species and a new Magnaporthiopsis species are proposed. TAXONOMY Pseudophialophora J. Luo & N. Zhang, gen. nov. MycoBank MB807080 Etymology: The generic name refers to the morphological similarity to Phialophora.
Conidiophores single or branched. Conidiogenous cells phialidic, curved, yellowish to hyaline. Conidia aggregated in slimy heads, oblong ellipsoidal, straight or 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 characteristics and formed a monophyletic clade in the phylogenetic tree. They were morphologically similar to Magnaporthiopsis and Gaeumannomyces in Phialophora-like conidial states and compressed hyphae in colonies on PDA. Magnaporthiopsis differed from them with faster growth rates, straight conidiogenous cells and wider and ovoid shaped conidia (Luo and Zhang 2013). Gaeumannomyces differed from them by having faster growing colonies, straight conidiogenous cells and presence of sickle-shaped conidia. This genus is also distinguishable from the true Phialophora, which usually has short and pigmented phialidic conidiogenous cells with a conspicuously flaring collarette, inhabiting rotten wood and potentially pathogenic to humans (Cole and Kendrick 1973, de Hoog et al. 1999, Gams 2000). Pseudophialophora is thus established. Pseudophialophora eragrostis J. Luo & N. Zhang, sp. nov. FIG. 2A–D MycoBank MB807081 Etymology: The specific epithet refers to the host’s generic name.
Colonies on PDA 2.7 cm diam after 7 d in the dark at 25 C, grass green, surface velvety to floccus, aerial mycelium yellowish, reverse pigmented, Cossack green. Colonies on CMA 1.6 cm after 7 d in the dark at 25 C, Schedes’s green, aerial mycelium sparse, reverse pigmented, peacock green. Conidiophores single or branched. Conidiogenous cells phialidic, curved, yellowish, 3–19 3 2–3.7 mm, 1.5–2.5 mm wide at the base, 0.5–1.2 mm wide near the apex (n 5 50). Conidia aggregated in slimy heads, oblong ellipsoidal,
M71
Lisa9 PRR1-4756
FF9
GI111 ATCC 44754
M47 M22
M23 M35
M51 M21
Ggt568
M55 CM2s8
73-15
GggFL64 GggFL199
M53 M54 CBS 235.32 R3-111a-1
CM7m9 CM9m11 CM10s2 ATCC 64411
Florida, USA
GgFL199
M33
Glenn County, California, USA Glenn County, California, USA
Kansas, USA Japan
unknown unknown
New Jersey, USA unknown
New Jersey, USA New Jersey, USA New Jersey, USA USA
Montana, USA New Jersey, USA
Florida, USA Florida, USA Arkansas, USA Washington, USA
the Netherlands
ATCC 38755
Host MCM7
RPB1
TEF1
JF710374
JF414896
JF710392
JF710442
JF710411
JF414895 JF414887
JF414883 JF414892
JF414885 JF414882
JF710389 JF710382
JF710384 JF710386
JF710390 JF710383
JF710440 JF710441
JF710432 JF710437
JF710433 JF710431
JF710417 JF710406
JF710408 JF710412
JF710415 JF710407
JX134659 JX134673 JX134685 JX134713 JX134727 JX134699
JX134658 JX134672 JX134684 JX134712 JX134726 JX134698
JF414846 JF414838
JF414834 JF414843
JF414836 JF414833
KF689595 KF689645 KF689635 KF689605 KF689615 KF689625 KF689596 KF689646 KF689636 KF689606 KF689616 KF689626 KF689594 KF689644 KF689634 KF689604 KF689614 KF689624 Genome data, Broad Institute
JF414875 JF414850 JF414900 JF710395 JF710445 JF710420 KF689593 KF689643b KF689633 KF689603 KF689613 KF689623
JF414872 JF414847 JF414897 JF710393 JF710443 JF710418 JF414873 JF414848 JF414898 JF710394 JF710444 JF710419 DQ341476 JX134669 JX134681 JX134709 JX134723 JX134695 Broad Institute
JF414871
JX134655 JX134668 JX134680 JX134708 JX134722 JX134694
DQ341473 JX134667 DQ341494 JX134707 JX134721 JX134693
Zoysia matrella JF414870 Oryza sativa JF414862
Oryza sp.
LSU
Genome data, Joint Genome Institute
Poa pratensis JF414858 unknown JF414867
Oryza sp.
ITS
DQ341471 JX134666 DQ341492 JX134706 JX134720 JX134692
SSU
Poa pratensis JF414860 unknown JF414857
Panicum sp. Panicum sp. Panicum sp. Triticum sp.
Triticum sp. Panicum sp.
Stenotaphrum secundatum unknown unknown Oryza sativa Genome data,
Avena sativa
Spartina alterniflora Connecticut, USA Castanea dentata UK Grass root
unknown
Source
CBS 187.65
CBS 610.75
EP155
N. oryzae
ATCC 22848
Isolate no.a Alternative no.
M69
Species
Species name, isolate number, alternative number, source, host and GenBank accession numbers of the fungi used in this study
Buergenerula spartinae Kohlm. & R.V. Gessner Cryphonectria parasitica (Murrill) M.E. Barr Gaeumannomyces cylindrosporus D. Hornby, Slope, Gutter. & Sivan. G. graminis var. avenae (E.M. Turner) Dennis G. graminis var. graminis (Sacc.) Arx & D.L. Olivier G. graminis var. graminis G. graminis var. graminis G. graminis var. graminis G. graminis var. tritici J. Walker G. graminis var. tritici Magnaporthiopsis panicorum J. Luo & N. Zhang M. panicorum M. panicorum M. panicorum M. poae (Landsch. & N. Jacks.) J. Luo & N. Zhang M. poae M. rhizophila (D.B. Scott & Deacon) J. Luo & N. Zhang M. rhizophila M. incrustans (Landsch. & N. Jacks.) J. Luo & N. Zhang M. incrustans Nakataea oryzae (Catt.) J. Luo & N. Zhang N. oryzae
TABLE I.
582 MYCOLOGIA
New Jersey, USA New Jersey, USA
New Jersey, USA CG-14 CG-4
CM9s6 AL3s4
AL2m1 M82 M83 70-15 M25 M60
Pyricularia grisea Sacc.
P. grisea
P. oryzae Cavara
P. oryzae P. oryzae
unknown New Jersey, USA
ITS
LSU MCM7
RPB1
TEF1
KF689601 KF689651 KF689641 KF689611 KF689621 KF689631 KF689600 KF689650 KF689640 KF689610 KF689620 KF689630
KF689597 KF689647 KF689637 KF689607 KF689617 KF689627 KF689602 KF689652 KF689642 KF689612 KF689622 KF689632
JF414839 JF414840
JF414888 JF414889
Genome data, Broad Institute JF710397 JF710398
JF710449 JF710447
JF710422 JF710423
JX134657 JX134671 JX134683 JX134711 JX134725 JX134697
Oryza sativa JF414863 Festuca JF414864 arundinacea
Oryza sativa
Digitaria sp.
Schizachyrium KF689599 KF689649 KF689639 KF689609 KF689619 KF689629 sp. Digitaria sp. JX134656 JX134670 JX134682 JX134710 JX134724 JX134696
Panicum sp. Poaceae sp.
Poaceae sp. Poaceae sp.
Eragrostis sp. KF689598 KF689648 KF689638 KF689608 KF689618 KF689628
JX134665 JX134679 JX134691 JX134719 JX134733 JX134705
JX134662 JX134676 JX134688 JX134716 JX134730 JX134702 JX134663 JX134677 JX134689 JX134717 JX134731 JX134703 JX134664 JX134678 JX134690 JX134718 JX134732 JX134704
a ATCC 5 American Type Culture Collection, Manassas, Virginia; CBS 5 Centraalbureau voor Schimmelcultures, Utrecht, the Netherlands; HKUCC 5 The University of Hong Kong Culture Collection, Hong Kong; YMF 5 the Yunnan Microbiological Fermentation Culture Collection Center, Yunnan, China. b Numbers in boldface indicate newly submitted sequences.
ATCC MYA4617 IG1, Pg rough 223S
New Jersey, USA New Jersey, USA
CM20m5-2 CM3m7
Tichnor, Arkansas, USA Tichnor, Arkansas, USA unknown
New Jersey, USA
YMF1.01288
M95
SSU
Panicum JX134660 JX134674 JX134686 JX134714 JX134728 JX134700 effusum var. effusum rotten wood JX134661 JX134675 JX134687 JX134715 JX134729 JX134701
Host
Yunnan, China rotten wood Hong Kong, China rotten wood Exeter, UK dead stem of dicot plant, probably Urtica dioica Yunnan, China rotten wood
CM12m9
YMF1.00981 HKUCC 3936 ATCC 24161
M92 CBS 114926 CBS 894.70
Yunnan, China
Queensland, Australia
Source
Pseudohalonectria lignicola Minoura & T. Muroi Pseudophialophora eragrostis J. Luo & N. Zhang P. eragrostis Pseudophialophora panicorum J. Luo & N. Zhang P. panicorum Pseudophialophora schizachyrii J. Luo & N. Zhang P. schizachyrii
YMF1.00980
M91
Ophioceras commune Shearer, J.L. Crane & W. Chen O. commune O. dolichostomum O. leptosporum (S.H. Iqbal) J. Walker
Isolate no.a Alternative no. ATCC 200212
Species
Continued
Omnidemptus affinis P.F. Cannon & Alcorn
TABLE I.
LUO ET AL.: NEW MAGNAPORTHACEAE SPECIES 583
584
MYCOLOGIA
FIG. 1. The maximum likelihood tree inferred from the combined SSU, ITS, LSU, MCM7, RPB1 and TEF1 sequence datasets. Branch values ($ 50%) of MP bootstrap proportions (MPBP) are noted above internodes. BI posterior probabilities (BIPP) $ 0.95 are shown as thickened branches.
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 of Eragrostis 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. Roots of Poaceae plant, 30 Aug 2012, J. Luo & N. Zhang CM20m5-2.
Notes: Two collections were clustered together and distinct from the other two species in genus Pseudophialophora. It is characterized by slow growing
LUO ET AL.: NEW MAGNAPORTHACEAE SPECIES
585
Poaceae plant, 30 Aug 2012, J. Luo & N. Zhang CM3m7 (HOLOTYPE, RUTPP-CM3m7). UNITED STATES. NEW JERSEY: Colliers Mills, N40 04.093, W74 26.598, 42 m. Roots of Panicum sp., 30 Aug 2012, J. Luo & N. Zhang CM9s6.
Notes: Two collections of this species occurred at the base of subclade F. Compared to Pseudophilophora eragrostis, they differed by having lighter colonies on PDA and Panicum host. Pseudophialophora schizachyrii J. Luo & N. Zhang, sp. nov. FIG. 2I–L MycoBank MB807083 Etymology: The specific epithet refers to the host generic name.
Colonies on PDA 2.2 cm diam after 7 d in the dark at 25 C, sulphine yellow, surface velvety, aerial mycelium yellowish, reverse pigmented, orange citrine. Colonies on CMA reaching 1.8 cm after 7 d in the dark at 25 C, aniline yellow, aerial mycelium sparse, reverse pigmented, pyrite yellow. Conidiophores single or branched. Conidiogenous cells phialidic, 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 wide near the apex (n 5 50). Conidia aggregated in slimy heads, oblong ellipsoidal to ellipsoidal, aseptate, hyaline, smooth, 5.5–8 3 2.5–3.5 mm (n 5 50). FIG. 2. Conidiophores and conidia. A–D. Pseudophialophora eragrostis. E–H. Pseudophialophora panicorum. I–L. Pseudophialophora schizachyrii. M–P. Magnaporthiopsis panicorum.
colonies on PDA and CMA, curved conidiophores cells, oblong ellipsoidal conidia and an endophytic nutrition strategy with Eragrostis grass host and assigned as the type species of the genus. Pseudophialophora panicorum J. Luo & N. Zhang, sp. nov. FIG. 2E–H MycoBank MB807082 Etymology: The specific epithet refers to the host generic name.
Colonies on PDA 2.6 cm diam after 7 d in the dark at 25 C, Javel green, surface velvety, aerial mycelium yellowish, reverse pigmented, oil green. Colonies on CMA 3.0 cm after 7 d in the dark at 25 C, pale greenyellow, aerial mycelium sparse, reverse pigmented, pale greenish yellow. Conidiophores single or branched. Conidiogenous cells phialidic, hyaline, curved, 4–22.5 3 1.5–2.7 mm, 1.2–2.2 mm wide at the base, 0.5–1.2 mm wide near the apex (n 5 50). Conidia aggregated 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
Specimens examined: UNITED STATES. NEW JERSEY: Assunpink Lake, N40 12.962, W74 30.527, 40 m. Roots of Poaceae plant, 30 Aug 2012, J. Luo & N. Zhang AL3s4 (HOLOTYPE, RUTPP-AL3s4). UNITED STATES. NEW JERSEY: Assunpink Lake, N40 12.962, W74 30.527, 40 m. Roots of Schizachyrium sp., 30 Aug 2012, J. Luo & N. Zhang AL2m1.
Notes: This species appeared to be more closely related to Pseudophilophora eragrostis than P. panicorum in the phylogenetic tree. Common characters of the genus could be found in all these species, however P. eragrostis differed from P. schizachyii by having green colonies, longer conidia and Eragrostis host. P. panicorum differed in greenish colonies, longer conidia and Panicum host. Magnaporthiopsis panicorum J. Luo & N. Zhang, sp. nov. FIG. 2M–P MycoBank MB807084 Etymology: The specific epithet refers to the host generic name.
Colonies on PDA 4.8 cm diam after 7 d in the dark at 25 C, parrot green, surface floccus, aerial mycelium yellowish, reverse pigmented, cedar green. Colonies on CMA reaching 5.5 cm after 7 d in the dark at 25 C, pale yellow-green, aerial mycelium sparse, reverse pigmented, pale yellow-green. Conidiophores single or branched. Conidiogenous cells phialidic, hyaline, straight or slightly curved, 5–30.5 3 2–3.5 mm 1.7–
586
MYCOLOGIA
2.5 mm wide at the base, 0.5–1.5 mm wide near the apex (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 of Panicum sp., 30 Aug 2012, J. Luo & N. Zhang CM2s8 (HOLOTYPE, RUTPP-CM2s8). UNITED STATES. NEW JERSEY: Colliers Mills, N40 04.093, W74 26.598, 42 m. Roots of Panicum sp., 30 Aug 2012, J. Luo & N. Zhang CM7m9. UNITED STATES. NEW JERSEY: Colliers Mills, N40 04.093, W74 26.598, 42 m. Roots of Panicum sp., 30 Aug 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. Zhang CM10s2.
Notes: Four collections of this species were grouped together and situated at the base of subclade C. Fast growing colonies with wavy and curling hyphae at the margin, phialidic conidiogenous cells, ovoid conidia and grass host made this species a good fit in Magnaporthiopsis (Luo and Zhang 2013). Compared to other species in the genus, M. poae differed from it by having olivaceous brown and faster growing colonies on PDA (1.3 cm/d at 28–30 C), and smaller conidia (3–8 3 1–3 mm) (Landschoot and Jackson 1989b). M. rhizophila differed by having gray-brown to olivacous black and faster growing colonies on PDA (0.8 cm/d at 28 C), wider conidiogenous cells and slightly longer conidia (6–20 3 2–6 mm) (Scott and Deacon 1983). M. incrustans differed by having olivaceous black and faster growing colonies on PDA (1.4 cm/d at 28–30 C), wider conidiogenous cells (9–15 mm) and smaller conidia (3–6 3 2–3 mm) (Landschoot and Jackson 1989a). DISCUSSION Studies have shown that there are three major lineages in Magnaporthaceae, a family in Sordariomycetes that includes more than 100 species (Cannon 1994, Luo and Zhang 2013). The early diverging lineage includes saprotrophic taxa that usually inhabit submerged woody substrates, such as Ophioceras and Pseudohalonectria Minoura & T. Muroi. The rice blast pathogen Pyricularia oryzae (Magnaporthe oryzae) and the gray leaf spot fungus Pyricularia grisea (Magnaporthe grisea) constitute the second lineage, which produce leaf-infecting sympodial conidia. More multilocus sequence data is needed to test whether other Pyricularia Sacc. species also belong to this lineage. The third lineage mainly is composed of grass root associated fungi, some of which are rootinfecting pathogens, such as Gaeumannomyces graminis (take-all pathogen of cereals) and Magnaporthiopsis poae (summer patch pathogen of turfgrass). The
asexual states of fungi in the third lineage are Phialophora-like or Harpophora-like, with the exception of Nakataea oryzae (Magnaporthe salvinii), which produces aerial infecting, sympodial conidia. The four new species proposed here belong in the third lineage, which is supported by their grass rootassociation habit, Phialophora-like conidial morphology and six-locus phylogenetic analysis. Eighty-three are names in Phialophora (http:// www.speciesfungorum.org/), which are poorly differentiated by morphology but highly divergent based on molecular phylogenetic analyses (Gams 2000, Harrington and McNew 2003, Vijaykrishna et al. 2004). The true Phialophora, including the type species P. verrucosa and its relatives, is linked to Capronia Sacc. in Herpotrichiellaceae of Chaetothyriales (Yan et al. 1995, Untereiner and Naveau 1999, de Hoog 1999). Based on phylogenetic studies (Gams 2000, Harrington and McNew 2003, Vijaykrishna et al. 2004, Thongkantha et al. 2009), some Phialophora species have been transferred or placed in other genera, such as Cadophora Lagerb. & Melin (Dermateaceae, Helotiales), Harpophora (Magnaporthaceae, Magnaporthales), Lecythophora Nannf. (Coniochaetaceae, 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 reported to have Phialophora-like or Harpophora-like asexual states (Cannon 1994, Re´blova´ 2006, Huhndorf et al. 2008, Zhang et al. 2011, Luo and Zhang 2013). Our six-gene phylogenetic analysis indicates that Magnaporthiopsis (with Phialophora-like anamorphs) is a sister genus of Gaeumannomyces graminis (with Harpophora anamorphs). Buergenerula spartinae, the only species with a Harpophora-like anamorph in the genus, appeared to be closely related to Magnaporthiopsis and Gaeumannomyces graminis. Gaeumannomyces cylindrosporus (with Harpophora anamorph) formed a distinct lineage. Ceratosphaeria lampadophora, the type species of the genus, and C. phialidica also produces Harpophora-like anamorphs (Re´blova´ 2006, Huhndorf et al. 2008). Previous LSU and SSU rDNA sequence analyses suggested that it was close to Pseudohalonectria (Huhndorf et al. 2008, Thongkantha et al. 2009). Taken together, species in Magnaporthaceae that produce Harpophora-like conidia are polyphyletic. The curved conidium morphology apparently evolved multiple times in this family. The three species in the proposed new genus Pseudophialophora formed a well supported monophyletic clade, with common features such as curved
LUO ET AL.: NEW MAGNAPORTHACEAE SPECIES conidiogenous cells without a conspicuous collarette at the apex that distinguish them from other Phialophora-like taxa. Many species in Magnaporthaceae are important pathogens of cereals and grasses (Scott and Deacon 1983; Landschoot and Jackson 1989a, b; Besi et al. 2009), but non-pathogenic members do not cause disease symptoms on their hosts. Studies have shown that nonpathogenic or weakly pathogenic Magnaporthaceae fungi can be used to suppress cereal diseases 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. graminis var. 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 did not cause disease symptoms on the grass hosts and might be of value in biological control of plant diseases. Greenhouse inoculations on rice, switchgrass and other plants are needed to test the pathogenicity of these fungi to further evaluate their nutritional mode and life cycle. ACKNOWLEDGMENT The research was financially supported by the National Science Foundation (grant DEB 1145174) to Zhang.
LITERATURE CITED Barnett HL, Hunter BB. 2006. Illustrated genera of imperfect fungi. 4th ed. St Paul, Minnesota: APS Press, 218 p. Besi MI, Tucker SL, Sesma A. 2009. Magnaporthe and its relatives. In: Encyclopedia of life sciences. Chichester, UK: John Wiley & Sons. p 1–9. doi: 10.1002/ 9780470015902.a0021311 Cain RF. 1952. Studies of fungi imperfecti I. Phialophora. Can J Bot 30:338–343, doi:10.1139/b52-025 Cannon PF. 1994. The newly recognized family Magnaporthaceae and its interrelationships. Syst Ascomycetum 13:25–42. Cole GT, Kendrick WB. 1973. Taxonomic studies of Phialiphora. Mycologia 65:661–688, doi:10.2307/ 3758266 de Hoog GS, Weenink XO, Gerrits van den Ende AHG. 1999. Taxonomy of the Phialophora verrucosa complex with the description of two new species. Stud Mycol 43:107–121. Deacon W. 1974. Interactions between varieties of Gaeumannomyces graminis and Phialophora radicicola on roots, stem bases and rhizomes of the Gramineae. Plant Pathol 23:85–92, doi:10.1111/j.1365-3059.1974.tb02914.x Gams W. 2000. Phialophora and some similar morphologically little-differentiated anamorphs of divergent ascomycetes. Stud Mycol 45:187–199.
587
Gutteridge RJ, Jankyn JF, Bateman GL. 2007. The potential of nonpathogenic Gaeumannomyces spp., occurring naturally or introduced into wheat crops or preceding crops, for controlling take-all in wheat. Ann Appl Biol 150:53–64, doi:10.1111/j.1744-7348.2006.00107.x Hall TA. 1999. Bioedit: a user-friendly biological sequences alignment editor analysis program for windows 95/98/ NT. Nucleic Acids Symp Ser 41:95–98. Harrington TC, McNew DL. 2003. Phylogenetic analysis places the Phialophora-like anamorph genus Cadophora in the Helotiales. Mycotaxon 87:141–151. Huhndorf SM, Greif M, Mugambi GK, Miller AN. 2008. Two new genera in the Magnaporthaceae, a new addition to Ceratosphaeria and two new species of Lentomitella. Mycologia 100:940–955, doi:10.3852/08-037 Landschoot PJ, Jackson N. 1989a. Gaeumannomyces incrustans sp. nov., a root-infecting hyphopodiate fungus from grass roots in the United States. Mycol Res 93:55– 58, doi:10.1016/S0953-7562(89)80136-4 ———, ———. 1989b. Magnaporthe poae sp. nov., a hyphopodiate fungus with a Phialophora anamorph from grass roots in the United States. Mycol Res 93:59– 62, doi:10.1016/S0953-7562(89)80137-6 Luo J, Zhang N. 2013. Magnaporthiopsis, a new genus in Magnaporthaceae. Mycologia 105:1019–1029, doi:10. 3852/12-359 Medlar EM. 1915. A new fungus, Phialophora verrucosa, pathogenic for man. Mycologia 7:200–203, doi:10. 2307/3753363 Nylander JAA. 2004. MrModeltest 2.2. Program distributed by the author. Uppsala, Sweden: Evolutionary Biology Centre. Re´blova´ M. 2006. Molecular systematics of Ceratostomella sensu lato and morphologically similar fungi. Mycologia 98:68–93, doi:10.3852/mycologia.98.1.68 Ridgway R. 1912. Color standards and color nomenclature. Washington DC: Ridgway. Ronquist F, Teslenko M, van der Mark P, Ayres D, Darling A, Ho¨hna S, Larget B, Liu L, Suchard MA, Huelsenbeck JP. 2012. MrBayes 3.2: efficient Bayesian phylogenetic inference and model choice across a large model space. Syst Biol 61:539–542, doi:10.1093/sysbio/sys029 Scott DB, Deacon JW. 1983. Magnaporthe rhizophila sp. nov., a dark mycelial fungus with a Phialophora conidial state, from cereal roots in South Africa. Trans Br Mycol Soc 81:77–81, doi:10.1016/S0007-1536(83)80206-X Slope DB, Salt GA, Broom EW, Gutterodge RJ. 1978. Occurrence of Phialophora radicicola var. graminicola and Gaeumannomyces graminis var. tritici on roots of wheat in field crops. Ann Appl Biol 88:239–246, doi:10.1111/j.1744-7348.1978.tb00701.x Speakman JB. 1984. Control of Gaeumannomyces graminis var. tritici in wheat by isolates of the Gaeumannomyces graminis var. graminis/Phialophora sp. (lobed hyphopodia) complex under field conditions. Phytopathol Z 109:188–191, doi:10.1111/j.1439-0434.1984.tb00706.x Swofford DL. 2002. PAUP* 4b10: phylogenetic analysis using parsimony (*and other methods). Sunderland, Massachusetts: Sinauer Associates. Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgin DG. 1997. The Clustal X Windows interface:
588
MYCOLOGIA
flexible strategies for multiple sequences alignment aided by quality analysis tools. Nucleic Acids Res 25: 4876–4882, doi:10.1093/nar/25.24.4876 Thongkantha S, Jeewon R, Vijaykrishna D, Lumyong S, Mckenzie EHC, Hyde KD. 2009. Molecular phylogeny of Magnaporthaceae (Sordariomycetes) with a new species Ophioceras chiangdaoense from Dracaena loureiroi in Thailand. Fungal Divers 34:157–173. Ulrich K, Augustin C, Werner A. 2000. Identification and characterization of a new group of root-colonizing fungi within the Gaeumannomyces-Phialophora complex. New Phytol 145:127–135, doi:10.1046/j.1469-8137.2000.00553.x Untereiner WA, Naveau FA. 1999. Molecular systematics of the Herpotrichiellaceae with an assessment of the phylogenetic position of Exophiala dermatitidis and
Phialophora americana. Mycologia 91:67–83, doi:10. 2307/3761194 Vijaykrishna D, Mostert L, Jeewon R, GamsW, Hyde KD, Crous PW. 2004. Pleurostomophora, an anamorph of Pleurostoma (Calosphaeriales), a new anamorph genus morphologically similar to Phialophora. Stud Mycol 50: 387–395. Yan ZH, Rogers SO, Wang CJK. 1995. Assessment of Phialophora species based on ribosomal DNA internal transcribed spacers and morphology. Mycologia 87:72– 83, doi:10.2307/3760949 Zhang N, Zhao S, Shen Q. 2011. A six-gene phylogeny reveals the evolution of mode of infection in the rice blast fungus and allied species. Mycologia 103:1267– 1276, doi:10.3852/11-022
